JP2008545614A - Phospholipid ether analogs for cancer detection and therapy - Google Patents

Abstract

The present invention uses a phospholipid ester analog in a subject of lung cancer, adrenal cancer, melanoma, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma, intestinal cancer A method for the treatment, detection of a relapsed cancer, a radiation and chemical insensitive cancer or a metastatic cancer selected from the group consisting of hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer and pancreatic cancer Provide methods and location identification methods.
[Selection] Figure 10

Description

Related applications

  This application claims priority from US Provisional Application No. 60 / 593,190 (filed December 20, 2004) and US Application 11 / ---- (filed December 20, 2005). Are all incorporated herein by reference for all purposes.

Background art

  The present invention relates generally to phospholipid ether analogs and uses thereof, and more particularly to the diagnosis of metastasis of various types of cancer (eg, non-small cell lung cancer, prostate cancer and those that have metastasized), It relates to the use of phospholipid ether analogs and combinations of these analogs for therapy, pharmacokinetics, dosimetry, and toxicity studies.

Non-small cell lung cancer (NSCLC)

  Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the United States today. Appropriately chosen patients are given the best chance for cure by surgical resection. Therefore, accurate preoperative assessment of local, regional and remote metastatic spread is important for optimal management.

  Due to improved sensitivity (especially when compared to CT imaging), imaging by FDG PET scanning has recently become the “gold standard” for NSCLC imaging. However, there is only about 90% sensitivity to distinguish mediastinal lymph node involvement and there is a lack of specificity (especially in patients with inflammatory or granulomatous disease) Especially a problem. Moreover, its use for diagnosing brain tumors or metastases to the brain is limited by the high metabolic background of normal brain tissue.

  Assessment of mediastinal lymph node status is essential because node metastasis, which occurs in nearly half of all patients with NSCLC, is probably the most frequent impediment to recovery. Accurate staging also prevents patients from undergoing unnecessary and incurable surgery. Therefore, there remains a need for imaging techniques that are more sensitive, specific, and accurate than any currently available technique.

  There is a limit to the current conventional treatment modality. Anatomical imaging with computed tomography (CT) and nuclear magnetic resonance imaging (MRI) is impractical for whole body screening, but is the most widely used non-invasive method for assessing the spread of local areas It is an invasive imaging method. However, CT relies on criteria of size greater than 1 centimeter to diagnose abnormal nodes.

Positron-emission tomography (PET) scanning using ¹⁸ F-fluorodeoxyglucose (FDG) has generated great interest as a tumor imaging technique. Recent studies have previously compared the standard approaches to staging NSCLC (CT, ultrasound, bone scan, etc.) and the ability of PET scans to detect metastases in the mediastinal lymph nodes and distant sites. Mediastinal complications were confirmed histopathologically and distant metastases were confirmed by other contrast studies. The sensitivity and specificity of PET detecting mediastinal metastases is 91% and 86%, respectively; the sensitivity and specificity of PET detecting remote metastases is 82% and 93%, respectively. This is compared with the sensitivity and specificity of CT scanning for mediastinal complications, 75% and 66%, respectively. A meta-analysis involving 39 studies and over 1000 patients also showed mediastinal staging (sensitivity and specificity were 85% and 90% for FDG-PET, respectively, 61% and 79% for CT scans) It was found that FDG-PET is more accurate than CT. However, when the CT showed enlarged mediastinal lymph nodes (78%), FDG-PET became less specific.

  It has been shown that FDG-PET reduces useless thoracotomy in patients. However, because of the false positive and false negative rates, mediastinoscopy for confirmation is often recommended. For example, a retrospective study involving more than 200 patients with NSCLC has shown FDG-PET sensitivity, specificity, positive and negative predictive value and accuracy of 64% and 77%, respectively. , 45%, 88% and 75%.

  FDG-PET also plays a role in diagnosing patients with extrathoracic disease, particularly in mid-stage lung cancer. A study conducted by the American Medical Association involving more than 300 patients revealed that 287 people had unexpected metastatic disease or second primary malignancy (sedonic primary malignancy) Of 18 patients (6.3%) were found to be identified. Some, but not all, suggests that by accurately identifying advanced disease, PET will avoid unnecessary thoracotomy in 1 in 5 patients.

  Conventional anatomical imaging techniques such as CT scanning are also not good at predicting survival after treatment. In a recent study involving 73 NSCLC patients receiving cisplatin-based chemo / radiotherapy at the same time or receiving radiotherapy alone, response with conventional CT imaging was not correlated with survival It was. However, the response by FDG-PET scanning was strongly correlated with survival (p <0.001). Survival from the day of follow-up PET scan was 84% and 84% at 1 year and 2 years, respectively, for 24 patients who responded completely with PET, but 32 patients who did not respond completely Were only 43% and 31%, respectively (p = 0.010). These results corroborate similar findings recently reported by other authors, correlating tumor biological malignancy with uptake on PET scans, and future survival of PET imaging long after treatment completion It was shown that the rate can be predicted at a high rate.

  It is generally accepted that FDG-PET imaging is a poor method for identifying brain metastatic disease in patients with NSCLC. Under normal circumstances, brain gray matter often uses glucose, and therefore FDG uptake is normally high. Cerebral metastatic disease is often very metabolic and often demonstrates increased FDG uptake, while frequently less than brain gray matter, and thus cerebral metastases may not be evident. In patients with NSCLC, the sensitivity and specificity to identify brain metastatic disease is 60% and 99% for FDG-PET and 100% and 100% for conventional imaging. Thus, FDG-PET imaging is not considered the best way to evaluate patients with NSCLC for metastatic disease to the brain.

  Another drawback of FDG is that it is not tumor specific and accumulates in both malignant and non-malignant hypermetabolic tissues. The overwhelming majority of false positive results with lung FDG-PET (positive results when radiation abnormalities are not due to cancer) are due to inflammatory and infectious factors. FDG is a non-specific tracer and accumulates in infected or inflamed areas. In the lung, these areas can be local lung parenchyma or more distinct areas (subsegmental, segmental or lobar) or hilar and mediastinal nodes. In a recent study in Japan, out of 116 lung nodules with a diameter of 1 to 3 cm, 15 of 73 malignant nodules were false negative with FDG-PET and 15 of 43 benign nodules were FDG- False positive with PET. In focal pneumonia causing ground glass opacities, the rate of false positives was as high as 80%. In another study, 10 patients with cancer outside the lung had false positive FDG-PET uptake in the lung; 6 had intense uptake in one lesion or multiple lesions. The person had a more regional or foliar uptake. In all 10 patients, uptake was due to sclerosis or atelectasis and the final diagnosis made later was lung inflammation or infection. In addition to active bacterial pneumonia, false positive FDG-PET results can occur in numerous other infectious and inflammatory conditions in the lung. In the Midwestern United States, many asymptomatic people have pulmonary nodules and enlarged nodes resulting from previous infection of histoplasma; many of these nodules are dormant but smoldering or active Some of them show infection in the wrong state. Pulmonary sarcoidosis is probably one of the more common active states of the inflammatory granulomas process in the lungs. Interestingly, in patients treated with oral corticosteroids for pulmonary sarcoidosis, FDG uptake decreased and then disappeared when consecutive FDG-PET scans were performed.

  FDG-PET is also frequently negative in malignant tumors with low metabolic rate (eg, bronchial cancer or carcinoid).

  Therefore, radiopharmaceuticals that can accurately identify early metastatic disease in patients with NSCLC have a significant impact on patient care, both in terms of staging and response to treatment. Let's go. In this area, PET imaging has improved diagnostic efficiency compared to CT, but it is not based on non-specific metabolic activity and is accurate based on a tumor-specific function that non-invasively sorts the entire body including the brain. There is still a need for new imaging techniques.

Prostate cancer

  In 2004 alone, approximately 230,110 new cases of prostate cancer will be diagnosed in the United States. Despite technical improvements in the most reliable local treatment of clinical organs restricted to prostate cancer by radiation prostatectomy, many men are treated with only the main treatment and follow-up ( follow-up) as many as 40% of patients will experience biochemical recurrence in the long term. This recurrence is typically defined as a post-operative PSA level of 0.4 ng / ml or greater. The reason for this is that patients with PSA levels above this threshold typically have clinical evidence of recurrence within 6-49 months. However, a PSA level of 0.2 or higher has been proposed more recently. Although results are limited, clinical and pathological diagnostic criteria are currently used to determine the likelihood of systemic disease recurrence. Factors that increase the likelihood of systemic recurrence include high preoperative PSA levels, as well as Gleason score> 7, seminal vesicle involvement and lymph node involvement, Pathological features of surgical specimens. In contrast, extra-articular dilation, clear surgical limits and Gleason score <7 are factors commonly associated with local recurrence. In addition, the rate of PSA elevation associated with prostatectomy has been used to determine whether the disease has recurred locally or systemically. For example, Partin et al. Reported that an increase in PSA of less than 0.75 ng / ml / year is more frequently associated with local recurrence. In addition, Patel et al. Reported that PSA doubling time exceeding 12 months correlates with local recurrence. Regardless of these clinical and pathological diagnostic criteria, the inventors are still unable to accurately select patients suitable for local treatment, resulting in many men having unnecessary ablation of hormones. I can receive it.

  One of the biggest challenges in treating patients with clinical organs limited to prostate cancer or patients with biochemical recurrence with limited treatment of diseases limited to organs deemed to be Still, to accurately distinguish local disease from metastatic disease. This diagnostic capability is important to identify patients who can benefit from an efficient local treatment treatment modality, including surgery, external beam irradiation, brachytherapy and cryotherapy. Inventors currently do not have an accurate method of staging, so patients with subclinical metastatic disease receive unnecessarily local treatment that leads to treatment risk factors. obtain. Moreover, patients with local recurrence who have elevated PSA and cannot reliably exclude systemic recurrence may experience hormonal ablation unnecessarily. This is not generally considered curable and is similar to the progression of prostate cancer independent of hormones, as well as progression of osteoporosis, loss of libido, weight gain, menopausal symptoms, and overall disability. Involved in pleasure.

  While conventional imaging studies such as computed tomography (CT) and nuclear magnetic resonance imaging (MRI) are useful in determining soft tissue metastases, the majority of prostate cancer metastasizes only to bone tissue. Therefore, the usefulness of CT and MRI scans in determining disease is suboptimal, and a more sensitive imaging treatment for either locally recurrent prostate cancer or metastatic prostate cancer Application style is required. Radioimmunoscintigraphy with indium-111 capromab pendide (ProstaScint, Cytogen Corp, Princeton, NJ) is a patient with prostate resection, elevated PSA, and inapparent in other contrast studies It has been used in patients with high clinical suspicion of metastatic disease and no clear evidence of metastatic disease. This scan is based on radiolabeled mouse monoclonal antibodies. This antibody is specific for PSMA (prostate specific membrane antibody) and is a transmembrane protein that is specifically expressed on both normal and malignant prostate epithelial cells. While ProstaScint radioimmunoscintigraphy has been shown to support a diagnosis of a disease that has recurred locally in the prostate bed in patients with elevated PSA, the clinical results of this scan have shown a range of sensitivity. The specificity is somewhat variable between 36% and 86%. Moreover, when subsequent biopsies are used as standard criteria for local recurrence, the false negative ProstaScint study reports 10% to 20% cases. In addition, false positive uptake of ProstaScint has been reported in neurofibromatosis, lymphoma, renal cancer, pelvic kidney, myolipoma and meningioma, and vertebrate bone marrow. According to this data, it is still controversial to use ProstaScint scans for patients at risk for occult metastases of prostate cancer.

In patients suffering from metastatic prostate cancer, positron emission tomography (PET) imaging has recently been used to measure the metabolic activity of bone metastases. This technique has proven to be effective in distinguishing between active bone metastases and osteoblast activity resulting from bone healing with successful treatment of metastatic disease. This can be better diagnosed by PET than either bone scan or CT. Moreover, changes in PET scan findings are the first to follow systemic treatment in patients with metastatic prostate cancer, whereas conventional bone scans often did not show significant changes. It can be seen in 4 weeks. Thus, the PET technique used for imaging can be useful for monitoring response to treatment in these patients. PET scanning with ¹⁸ F-FDG has generated significant benefits as an imaging technique. Recently, FDG-PET has been shown to be able to distinguish between active and stationary bone metastases in patients with prostate cancer. The intensity of FDG uptake is thought to reflect the metabolic and biological activity of these lesions, in contrast, conventional bone scans using technetium-diphosphonate compounds exhibit nonspecific osteoblast activity. , Can be detected as a false positive sign associated with treatment. In addition, early metastases initially seeded in the bone marrow do not necessarily give signs until an osteoblastic response occurs, so a false negative interpretation can be obtained. Thus, a persistently positive bone scan does not necessarily indicate the presence of remaining viable metastases, and a negative bone scan result accurately identifies the patient's metastatic tumor burden. Cannot be reflected. FDG-PET can therefore prove to be beneficial in guiding the management of patients with bone metastases, and in retrospective studies, FDG-PET and helical CT are useful in detecting metastatic disease. It has been independently shown to be more efficient than ¹¹¹ In-monoclonal antibody imaging.

  While FDG-PET scanning is a promising imaging technique in patients with prostate cancer, most prostate cancers grow slowly and therefore do not accumulate FDG. Therefore, it is not well imaged with the drug. Moreover, FDG is excreted in the urine, and the accumulation of FDG in the bladder minimizes the possibility of detecting local recurrence of prostate cancer. In fact, Morris et al. Report that detection of parenchyma metastasis by FDG-PET alone is difficult if the metastatic site is obscured by the anatomical route of tracer excretion. More recently, PET-CT is known to be more efficient than PET alone in identifying metastatic foci in patients with suspicious but occult metastases. In an expected study on patients with various tumor types, the specificity and accuracy associated with the interpretation of many radioactive materials was significantly higher with PET-CT.

  Therefore, there is a need to develop more sensitive and specific imaging tests, molecular contrast agents (eg phospholipid ether compounds (PLE)). Tumor selection that minimally accumulates in the bladder, accurately identifies early metastatic disease in patients with prostate cancer, and has a significant impact on patient care in terms of both staging and response to treatment It is desirable to have an active radiopharmaceutical.

Summary of the present invention

  The present invention relates to cancer that has recurred, cancer that is insensitive to radiation and chemicals, or cancer that has metastasized in subjects with cancer or suspected of having cancer (these cancers are lung cancer, adrenal cancer, melanoma). , Colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma, intestinal cancer, hepatocellular carcinoma, retinoblastoma, cervical cancer ), Selected from the group consisting of glioma, breast cancer and pancreatic cancer) and provides a method for identifying the location. The method comprises the following steps: (a) administering a phospholipid ether analog to the subject; and (b) having a cancer that has recurred, is insensitive to radiation and chemicals, or has metastasized in the subject. It is then determined whether the suspected organ retains a higher level of analog than the surrounding area (s). Here, the higher residence area suggests the detection and location of cancer that has recurred, cancer that is insensitive to radiation and chemicals, or that has metastasized.

  In a more preferred embodiment, the phospholipid analog is:

Wherein X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent, or

Where X is a radioisotope 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 NH ₂ , NR ₂ and NR ₃ , wherein R is an alkyl or arylalkyl substituent;
More selected. Further, in certain embodiments, X is a halogen radioisotope consisting of ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I and ²¹¹ At. Selected from the group. More preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propanediol-3-phosphocholine, or 1 -O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, where iodine is a radioisotope form. In this method, detection is preferably performed by PET, CT, MRI scanning methods and combinations thereof.

  Another embodiment of the invention provides a method of treating cancer that has recurred, cancer insensitive to radiation and chemicals, or cancer that has metastasized in a subject. The method includes administering to the subject an effective amount of a compound comprising a phospholipid ether analog. In preferred embodiments, the cancer that has recurred, cancer that is insensitive to radiation and chemicals, or cancer that has metastasized is lung cancer, adrenal cancer, melanoma, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer. Occurring in a group selected from squamous cell carcinoma, intestinal cancer, hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer and carcinosarcoma. Also preferably, the phospholipid analog is:

Wherein X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent, or

Where X is a radioisotope 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 NH ₂ , NR ₂ and NR ₃ , wherein R is more selected as an alkyl or arylalkyl substituent. Preferably, in this method, X is a halogen radioactivity comprising ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At and combinations thereof. Selected from the group of isotopes. More preferably, the effective amount of the phospholipid ether analog is a combination of at least two isotopes, one having a path range of about 0.1 mm to 1 mm and the other having an orbit range of 1 mm to 1 m.

Most preferably, the effective amount of the phospholipid ether analog is a combination of at least two isotopes ¹²⁵ I and ¹³¹ I. Similarly, preferably the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propanediol-3-phosphocholine, Or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, where iodine is in the form of a radioisotope.

  In certain embodiments, the effective amount of phospholipid ether analog is divided. In still other embodiments, an effective amount of the phospholipid ether analog can be processed in a linear and dose dependent manner from about 0.5 μCi to about 3 Ci. Other embodiments provide that the dose is adjustable with respect to the volume of the cancer. Yet another embodiment provides that the dosage for radiation insensitive tumors is greater than the dosage for radiation sensitive tumors and less than 3 Ci and can be adjusted for the volume of the cancer. To do.

  Another embodiment of the present invention provides the use of a phospholipid ether analog for the manufacture of a pharmaceutical composition for treating recurrent cancer, cancer insensitive to radiation and chemicals, metastasized cancer. In this embodiment, the phospholipid analog is:

Where X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent, or

Where X is a radioisotope 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 NH ₂ , NR ₂ and NR ₃ , wherein R is more selected as an alkyl or arylalkyl substituent. Also in this embodiment, preferably X is from ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At and combinations thereof Selected from the group of radioactive halogen isotopes. Also preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propanediol-3-phosphocholine, or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, iodine is a radioisotope form.

  Further objects, features and advantages of the present invention will become apparent from the following detailed description, drawings and appended claims.

Detailed Description of the Invention

  DETAILED DESCRIPTION OF THE INVENTION Prior to describing the method of the present invention, the present invention is not limited to the specific methodologies, protocols, cell lines and reagents described herein, which may be varied. Is understood. The terminology used herein is used for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined by the appended claims. It should also be understood that it is limited only by the scope.

  As used herein and in the appended claims, the singular forms “a”, “an” and “the” are used herein to refer to It should be noted that the plural is included unless otherwise specified. Thus, for example, reference to “a cell” includes a plurality of such cells and equivalents known to those skilled in the art and others. Similarly, “one (a)” (or “an”), “one or more” and “at least one” may be used interchangeably herein. It should also be noted that the terms “comprising”, “including” and “having” may be used interchangeably.

  Unless defined otherwise, all technical and natural science terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and the more preferred methods and materials are now described. For purposes of describing and disclosing chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies (which are reported in the above publications) that may be used in connection with the present invention, All publications mentioned in the above are incorporated herein by reference. Anything not described in this specification should be construed as an understanding that the invention is not described with the expectation of the description according to the prior invention.

  As defined herein, the term “isomer” refers to an optical isomer and its analogs, a structural nucleoisomer and its analogs, a conformational isomer. ) And analogs thereof, but not limited thereto. In one embodiment, the invention encompasses the use of different optical isomers of the anti-tumor compound of formula 3A. Those skilled in the art will appreciate that anti-tumor compounds useful in the present invention may contain at least one chiral center. Thus, the compounds used in the methods of the invention exist in optically active or racemic forms and can be isolated. Compounds that exhibit polymorphism may also exist.

  It is to be understood that the present invention can encompass the use of any racemic, optically active, polymorphic, or stereoisomeric form or mixtures thereof. These forms have the characteristic of being useful for the treatment of tumor-related conditions as described herein and in the claims. In one embodiment, the anti-tumor compound may comprise a pure (R) -isomer. In another embodiment, the anti-tumor compound may comprise a pure (S) -isomer. In another embodiment, the compound may comprise a mixture of (R) and (S) isomers. In another embodiment, the compound may comprise a racemic mixture comprising both the (R) -isomer and the (S) -isomer. Isolating optically active forms chromatographically using chiral stationary phases, by chiral synthesis, by synthesis from optically active starting materials, eg by dissolving the racemate by recrystallization techniques Are well known to those skilled in the art.

  The present invention encompasses the use of pharmaceutically acceptable salts of compounds amino substituted with organic and inorganic acids such as citric acid and hydrochloric acid. The present invention also includes N-oxides of the amino substituents of the compounds described herein. Pharmaceutically acceptable salts can also be prepared from phenolic compounds treated with an inorganic base such as sodium hydroxide. Similarly, esters of phenolic compounds can be made with aliphatic and aromatic carboxylic acids such as acetates and benzoates. As used herein, the term “pharmaceutically acceptable salt” refers to a compound that achieves substantially the same pharmaceutical effect as the basic compound and is formulated from the basic compound.

  The present invention further includes a method utilizing a derivative of the antitumor compound. The term “derivative” includes, but is not limited to, ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. Moreover, the present invention further includes a method utilizing the hydrate of the anti-tumor compound. The term “hydrate” includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.

  The present invention further includes a method of using a metabolite of the antitumor compound. The term “metabolite” means any substance produced from another substance by metabolic action or a metabolic process.

  As defined herein, “contact” means that the anti-tumor compound used in the present invention is a test tube, a flask, a tissue culture, a chip, an array, a plate, Meaning incubated in a sample containing the receptor in a microplate, capillary, etc. and incubated for a sufficient time at a temperature sufficient to allow the anti-tumor compound to bind to the receptor. Methods of contacting the sample with the anti-tumor compound or other specific binding compound are known to those of skill in the art and can be selected depending on the type of assay protocol being performed. Incubation methods are also standard and 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 to be treated and the compound is contacted in vivo. Means you can.

  As used herein, the term “treating” encompasses prophylaxis as well as disorder remission treatment. As used herein, the terms “reducing”, “suppressing”, and “inhibiting” are lessening or decresing. ) Having a generally understood meaning. As used herein, the term “progression” means that the range or severity increases, progresses, grows, and worsens. As used herein, the term “relapse” means re-affected by the disease after remission.

  As used herein, the term “administering” refers to contacting a patient, tissue, organ, or cell with an anti-tumor phospholipid ether compound. As used herein, administration can be performed in vitro (ie, in a test tube) or in vivo (ie, in a cell or tissue of a living organism (eg, a human)). In certain embodiments, the invention includes administering a compound useful in the invention to a patient or subject. “Patient” or “subject” is used herein equivalently and refers to a mammal, preferably a human, that is any of the following: (1) Administration of an anti-tumor substance using a phospholipid ether compound The disorder can be ameliorated or treatable, or (2) susceptible to a disorder that can be prevented by administration of an antitumor compound using a phospholipid ether compound.

  As used herein, a “pharmaceutical composition” refers to a therapeutically effective amount of an anti-tumor compound (which includes radioactivity and appropriate diluents, preservatives, solubilizers, emulsifiers and emulsifiers). Adjuvants, ie, those collectively referred to as “pharmaceutically acceptable carriers”. As used herein, the terms “effective amount” and “therapeutically effective amount” produce the desired therapeutic response without undue side effects (eg, toxic, inflammatory or allergic reactions). Refers to the amount of therapeutic agent that is sufficiently active. The specific “effective amount” clearly depends on the particular situation being treated, the physical condition of the patient, the type of animal being treated, the duration of treatment, the nature of the co-treatment (if any), as well as the particular used It will vary depending on factors such as the formulation and the structure of the compound or derivative thereof. In this case, the amount is considered therapeutic if provided with one or more of the following: (a) prevention of the disease (eg pancreatic cancer, breast cancer); and (b) reversal of such disease. Or stabilization. The optimal effective amount can be readily determined by one of ordinary skill in the art using routine experimentation.

  The pharmaceutical composition is a liquid formulation, or a lyophilized formulation or otherwise dried formulation, and various buffer diluents (eg, Tris-HCI, acetate, Phosphates), pH and ionic strength, additives (eg, albumin or gelatin that inhibits absorption to the surface), surfactants (eg, Tween 20 (polysorbate) 20, Tween 80, Pluronic F68, bile salts) , Solubilizers (eg glycerol, polyethylene glycerol), antioxidants (eg ascorbic acid, sodium metabisulfite), preservatives (eg thimerosal, benzyl alcohol, parabens), bulking substances or isotonic Tonicity modifier (for example, , Lactose, mannitol), covalent bonding of polymers (eg, polyethylene glycol to proteins), complex formation with metal ions, or polymerizable compounds (eg, polylactic acid, polyglycolic acid, hydrogel, etc.) Incorporation of the substance in or on a microparticulate formulation or onto a liposome, microemulsion, micelle, monolayer or multilamellar vesicle, erythrocyte ghost, or spheroplast. Such compositions will affect the physical state, solubility, stability, release rate in vivo, and clearance rate in vivo. Compositions whose release is controlled or maintained are included in the formulation in a fat-soluble long-acting drug (eg, fatty acids, waxes, oils).

  Similarly, encompassed by the present invention is a method of administering a particulate composition coated with a polymer (eg, poloxamer or poloxamines). Other embodiments of this composition include microparticles that form protective coatings, protease inhibitors or permeation enhancers for a variety of administration routes, including topical administration, parenteral administration, pulmonary administration, intranasal administration and oral administration. Incorporate. In certain embodiments, the pharmaceutical composition is administered parenterally, near-cancerous, mucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, Intracranial and intratumoral administration.

  Further, as used herein, “pharmaceutically acceptable carriers” are well known to those skilled in the art and include 0.01-0.1M and preferably 0.05M phosphorous. Examples include, but are not limited to, acid buffer or 0.9% saline. Moreover, such pharmaceutically acceptable carriers can be aqueous solutions, non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (eg 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 solution, dextrose and sodium chloride, lactated Ringer and non-volatile oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (eg, based on Ringer's glucose solution). Preservatives and other additives (eg, antibiotics, antioxidants, collating agents, inert gases, etc.) may also be present.

  The administrable, controlled or sustained release compositions of the present invention include formulations in fat soluble depots (eg fatty acids, waxes, oils). Similarly, encompassed by the present invention are particulate compositions coated with a polymer (eg, poloxamer or poloxamine) and compounds conjugated to antibodies directed against tissue specific receptors, ligands or antigens, Alternatively, a method of administering a compound bound to a ligand of a tissue specific receptor.

  Other embodiments of compositions administered in accordance with the present invention include microparticulate forms, protective coatings, protease inhibition for a variety of administration routes, including parenteral, pulmonary, intranasal and oral administration. Agent or permeation enhancer.

  A compound modified by a covalent attachment of a water-soluble polymer (eg, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone or polyproline) is the corresponding unmodified compound It is known to exhibit a substantially longer half-life in blood after intravenous injection (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications can increase the solubility of the compound in aqueous solution, eliminate aggregation, increase the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound Can be. As a result, the desired biological activity in vivo is achieved by the abduction of such polymeric compounds, with less frequent administration than unmodified compounds or less doses than unmodified compounds. obtain.

  In yet another method of the invention, the pharmaceutical composition can be delivered in a controlled release system. For example, the agent can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes or other modes of administration. In one embodiment, a pump can be used (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used. In yet another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target (eg, the liver), and thus only a small portion of the systemic dose is administered (eg, Goodson, In Medical Applications of Controlled). Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in a review by Langer (Science 249: 1527-1533 (1990)).

  The pharmaceutical formulation may contain only the anti-tumor compound, or may further contain a pharmaceutically acceptable carrier, and is in a solid or liquid form (eg, tablets, powders, capsules, implants) ), Aqueous solutions, suspensions, elixirs, emulsions, gels, creams or suppositories (including rectal suppositories and urethral suppositories)). Pharmaceutically acceptable carriers include gums, starches, sugars, cellulosic materials and mixtures thereof. A pharmaceutical formulation containing an anti-tumor compound can be administered to a patient, for example, by implantation of an implanted tablet subcutaneously. In a further embodiment, the implant provides a controlled release of the anti-tumor compound over an extended period of time. The formulation can also be administered by intravenous, arterial or intramuscular injection of liquid or solid formulations or by topical application. Administration can also be accomplished by the use of rectal suppositories or urethral suppositories.

  The pharmaceutical preparations that can be administered according to the invention can be prepared by known dissolution, mixing, granulating or tablet-forming processes. Anti-tumor compounds or physiologically acceptable derivatives thereof (eg, salts, esters, N-oxides, etc.) for oral administration are conventional additives (eg, vehicles, stabilizers or inert dilutions) for this purpose. And converted to a form suitable for administration (eg, tablet, coated tablet, hard gelatin capsule or soft gelatin capsule, aqueous solution, alcoholic solution or oily solution) by conventional methods. Examples of suitable inert vehicles are in combination with binders (eg acacia, corn starch, gelatin), in combination with disintegrants (eg corn starch, potato starch, alginic acid) or lubricants (eg stearic acid). Or a conventional tablet base (eg lactose, sucrose or corn starch) in combination with magnesium stearate).

  Examples of suitable oily vehicles or solvents are vegetable oils or animal oils (eg sunflower oil or fish-liver oil). The formulation can be achieved both as dry granules and as wet granules. Parenteral administration (subcutaneous injection, intravenous injection, arterial injection or intramuscular injection) if a suitable conventional substance for this purpose is desired (eg, solubilizers or other auxiliaries) Antitumor compounds or physiologically acceptable derivatives (eg salts, esters, N-oxides, etc.) are converted into solutions, suspensions or emulsifiers. Examples are sterile liquids (eg water and oils) with or without the addition of surfactants and other pharmaceutically acceptable adjuvants. Exemplary oils are sterile liquids of petroleum, animal oils, vegetable oils or oils of synthetic origin (eg, peanut oil, soybean oil or mineral oil). In general, water, saline, aqueous dextrose and similar sugar solutions, and glycols (eg, propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions. It is.

  The preparation of a pharmaceutical composition that contains an active ingredient is well known in the art. Such compositions can be prepared as aerosols delivered to the nasopharynx or can be prepared as injectables, either as solutions or suspensions; however, solutions prior to injection Solid forms suitable for placement in or in suspension can also be prepared. The formulation 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 and the like or combinations thereof.

  In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the effectiveness of the active ingredient.

  The active ingredient can be formulated into a composition in the form of a neutralized pharmaceutically acceptable salt. Pharmaceutically acceptable salts include acid addition salts, which are formed with inorganic acids (eg, hydrochloric acid or phosphoric acid) or organic acids (eg, acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Can be mentioned. Salts formed from free carboxyl groups are also converted to inorganic bases (eg sodium, potassium, ammonium, calcium or ferric hydroxide) and organic bases (isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.). Can come from.

  For example, the antitumor compounds or physiologically acceptable derivatives (eg salts, esters, N-oxides, etc.) are prepared for topical administration on the body surface using creams, gels, drops, etc. Applied as a solution, suspension or emulsifier in a physiologically acceptable diluent with or without a carrier.

  In another method according to the invention, the active compound can be delivered in the form of vesicles, in particular liposomes (see Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious. Disease and Cancer, Lopez-Berstein and Fiddler (eds.), Liss, NY, pp. 353-365 (1989); Lopez-Berstein ibid., Pp. 317-327;

  For use in medicine, the anti-tumor compound salt may be a pharmaceutically acceptable salt. However, other salts may be useful in the preparation of the compounds of the invention or the preparation of these pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compound include acid addition salts, which include, for example, a solution of the compound of the invention and a pharmaceutically acceptable acid (eg, hydrochloric acid, sulfuric acid). ), Methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carboxylic acid or phosphoric acid).

In general, phospholipid ether compounds, particularly NM404, are promising new tumor-selective diagnostic contrast agents for monitoring the therapeutic response of several tumor therapy therapeutic applications. Radioiodinated NM404 is a second generation phospholipid ether analog but showed significant tumor selectivity in a 27/27 tumor model. A promising hypothesis of this approach, due to the lack of metabolic phospholipase enzyme in the plasma membrane of tumor cells, is that phospholipid ether analogs have been collected exclusively at the tumor cell membrane because they cannot be metabolized and excreted. It is. Thus, the difference in the clearance rate of phospholipid ether from normal cells versus the clearance rate of phospholipid ether from living tumor cells forms the basis of this concept. The results obtained in various tumor models show that NM404 is captured, selectively retained in viable tumor cells, and the primary and metastatic lesions, regardless of the anatomical site including the lesions found in the lymph nodes. Suggests that it is localized to both. Unlike FDG, this drug is not localized at the site of infection. Other advantages of NM404 over FDG include: FDG is not selective for tumor cells, but migrates to the site of infection and thickens (Barrett's esophagus), whereas NM404 is a malignant tumor cell. It is selective for and stays indefinitely in malignant tumor cells. In addition, FDG has a half-life of 110 minutes and may be limited in delivery within 200 miles of the production point, but ¹²⁴ I has a physical half-life of 4 days, so it can be transported anywhere in the world. Whereas FDG does not have any therapeutic capacity, NM404 stays long (not metabolized) and therefore, when combined with a suitable radioisotope such as ¹³¹ I, NM404 produces significant therapeutic capacity . NM404 can be labeled with a variety of iodine isotopes, which expands the versatility of NM404 (diagnostic and therapeutic, and tools for laboratory animal research), while FDG is ¹⁸ F for PET scanning. Or potentially limited to ¹⁹ F (stable) for nuclear magnetic resonance imaging, despite very low sensitivity. Despite its ability to target tumors, FDG has no therapeutic potential because of its rapid metabolism in tumor cells. NM404 not only accurately predicts local tumor response to various therapeutic regimens, but also enables detection of distant metastatic lesions when treating primary tumors at sub-therapeutic doses To do.

The PLE compounds can be designed to more accurately estimate the specificity and sensitivity of radiolabeled PLE analogs (eg, NM404) as contrast agents in prostate cancer and other cancers. Based on preclinical models, PLE analogs such as NM404 tend to show high uptake in tumors, and because of this trend, this drug has significant potential for use in staging, response following treatment. And potentially therapeutically effective alpha-emitting halogens can be used as therapeutic agents when combined with high doses of ¹³¹ I, ¹²⁵ I or ²¹¹ At.

Preferred embodiment

The present invention generally provides methods and techniques for the detection and treatment of various cancers. The present invention relates to cancers that have recurred, cancer that is insensitive to radiation and chemicals, and cancer that has metastasized (subjects of lung cancer, adrenal cancer, melanoma, colon cancer) , Colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma, intestinal cancer, hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer and pancreatic cancer ) And provide a method for determining the position. The method comprises the following steps: (a) administering a phospholipid ether analog to the subject; and (b) a cancer that has recurred, is not sensitive to radiation, or has metastasized in the subject. Determining whether an organ suspected of having retains a higher level of analog than the surrounding region (s). Here, the higher residence area indicates the detection and location of cancer that has recurred, cancer that is insensitive to radiation and chemicals, or cancer that has metastasized.

  In a more preferred embodiment, the phospholipid analog is:

Where X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent.
Or

Where X is a radioisotope 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 NH ₂ , selected from the group consisting of NR ₂ and NR ₃ , wherein R is more selected as an alkyl or arylalkyl substituent. Further, in certain embodiments, X is a radioactive halogen isotope consisting of ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I and ²¹¹ At. Selected from the group. More preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propanediol-3-phosphocholine, or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, iodine is a radioactive isotope form. In this method, detection is preferably performed by a PET scanning method, a CT scanning method, an MRI scanning method, and a combination thereof.

As used herein, an “alkyl” group refers to a straight, branched or cyclic, saturated or unsaturated aliphatic hydrocarbon. The alkyl group has 1 to 16 carbons and may be unsubstituted or halogen, hydroxy, alkoxy, carbonyl, amide, alkylamide, dialkylamide, nitro, amino, alkylamino, dialkylamino, carboxyl , Optionally substituted by one or more groups selected from thio and thioalkyl. A “hydroxy” group refers to an OH group. An “alkoxy” group refers to an —O-alkyl group, where alkyl is as defined above. A “thio” group refers to a —SH group. A “thioalkyl” group refers to a —SR group, where R is alkyl as defined above. An “amino” group refers to a —NH ₂ group. An “alkylamino” group refers to a —NHR group, where R is alkyl as defined above. A “dialkylamino” group refers to a —NRR ′ group, where R and R ′ are all as defined above. An “amido” group refers to —CONH ₂ . An “alkylamido” group refers to a —CONHR group, wherein R is alkyl as defined above. A “dialkylamido” group refers to a —CONRR ′ group, where R and R ′ are alkyl as defined above. A “nitro” group refers to a NO ₂ group. A “carboxy” group refers to a COOH group.

  As used herein, “aryl” includes both carbocyclic and heterocyclic aromatic rings, both monocyclic and fused polycyclic, wherein the aromatic ring is It can be a 5-membered ring or a 6-membered ring. Representative monocyclic aryl groups include, but are not limited to, phenyl, furanyl, pyrrolyl, thienyl, pyridinyl, pyrimidinyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl and the like. A fused polycyclic aryl group is an aromatic group that includes a 5-membered or 6-membered aromatic ring or aromatic heterocycle as one or more rings in the fused ring system. Representative fused polycyclic aryl groups include naphthalene, anthracene, indolizine, indole, isoindole, benzofuran, benzothiophene, indazole, benzimidazole, benzothiazole, purine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline. Quinoxaline, 1,8-naphthyridine, pteridine, carbazole, acridine, phenazine, phenothiazine, phenoxazine, and azulene. Further, as used herein, “arylalkyl” refers to a moiety (eg, benzyl) in which an aromatic is linked to an alkyl group attached to the site indicated in the PLE compound. .

  Another embodiment of the invention provides a method for the treatment of recurrent cancer, radiation and chemical insensitive cancer or metastasized cancer in a subject. The method includes administering to the subject an effective amount of a compound comprising a phospholipid ether analog. In preferred embodiments, the cancer that has recurred, cancer that is insensitive to radiation and chemicals, or cancer that has metastasized is lung cancer, adrenal cancer, melanoma, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, It occurs in a group selected from squamous cell carcinoma, intestinal cancer, hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer and carcinosarcoma. Also preferably, the phospholipid analog is:

Where X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent. Or

Where X is a radioisotope of halogen; n is an integer between 8 and 30; Y is selected from the group consisting of H, OH, COOH, COOR and OR; selected from the group consisting of NH _2, NR ₂ and NR _3, wherein, R is a substituent of alkyl or arylalkyl, it is selected from. In this method, preferably X is a radioactive halogen consisting of ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At and combinations thereof. Selected from the group of isotopes. More preferably, the effective amount of the phospholipid ether analog is a combination of at least two isotopes, one as described in FIG. 2, one orbital range of about 0.1 mm to 1 mm, and the other about The trajectory range is 1 mm to 1 m.

Most preferably, the effective amount of the phospholipid ether analog is a combination of at least two isotopes, ¹²⁵ I and ¹³¹ I. Similarly, preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propanediol-3-phosphocholine. Or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, where iodine is in the form of a radioisotope.

  In certain embodiments, as shown in FIG. 11, an effective amount of a phospholipid ether analog is resolved. The advantage of using split doses is that this allows the PLE analog to be removed from normal tissue. As in the examples, divided doses of NM404 produced the same therapeutic effect (eg, 3 × 50 microCi vs. a single dose of 150 microCi), while lower doses of compound (which were then split Further excluded from normal tissue between injections and split injections).

  In yet other embodiments, the effective amount of the phospholipid ether analog is from about 0.5 μCi to about 500 mCi, which can be treated in a linear and dose-dependent manner, as shown in FIG. . Other embodiments provide that the dosage is adjustable with respect to the volume of the cancer as shown in FIG. The graph in FIG. 12 shows the difference in tumor growth for the same dose of I-125-NM404 (150 μCi) when injected into animals with small tumors (<1 cm) and large tumors (> 1 cm).

  The results showed that for small tumors the tumor growth was overwhelmed at the dose, but the dose did not show any effect on the large tumor population, i.e. basically a non-irradiated control. It shows that it behaved like. Further demonstrated in the graph of FIG. 12 is that the effective dose to the tumor should be adjusted to the volume of the tumor and the tumor per volume of tumor volume to be achieved to show efficacy There is a dose.

  Still other embodiments provide for radiation doses to tumors that are insensitive to radiation and chemicals, as confirmed by comparing the PC3 cancer model (FIG. 8) and the A549 cancer model (FIGS. 10, 11 and 12). It provides more than the dose for sensitive tumors, less than 500 mCi, and can be adjusted to the volume of the cancer. Here, the PC3 model is radiation insensitive.

  Another embodiment of the present invention provides the use of a phospholipid ether analog to produce a pharmaceutical composition for the treatment of recurrent cancer, radiation and chemical insensitive cancer or metastasized cancer. In this embodiment, the phospholipid analog is:

Where X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent, or

Where X is a radioisotope of halogen; n is an integer between 8 and 30; Y is selected from the group consisting of H, OH, COOH, COOR and OR; selected from the group consisting of NH _2, NR ₂ and NR _3, wherein, R is a substituent of alkyl or arylalkyl, it is selected from. Similarly in this embodiment, preferably, X is from ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At and combinations thereof Is selected from the group of radioactive halogen isotopes. Also preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propanediol-3-phosphocholine, Or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, where iodine is in the form of a radioisotope.

Effects of synthesis and structure-activity relationships on tumor avidity of radioiodinated phospholipid ether analogues

  Radioiodinated phospholipid ether analogs show a remarkable ability to accumulate selectively in various human and animal tumors in xenografts and in rodent models with spontaneous tumors. This tumor avidity is believed to arise as a result of metabolic differences between the tumor and the corresponding normal tissue. The results of this study indicate that one factor in the tumor retention of these compounds in the tumor is the length of the alkyl chain that determines the hydrophobic nature. By reducing the chain length from C12 to C7, the compound caused little or no tumor accumulation within 24 hours of administration and resulted in rapid clearance of the compound in tumor-bearing rats. . Increasing chain length produced the opposite effect, with analogs of C15 and C18 delaying plasma clearance and showing increased tumor uptake and retention in tumor-bearing rats. Tumor uptake exhibited by the propanediol analogs NM-412 and NM-413 was associated with high levels of liver and abdominal radioactivity 24 hours after injection into tumor-bearing rats. Adding a 2-O-methyl moiety to the propanediol backbone also significantly delayed tumor uptake. In human PC-3 tumor-bearing immunodeficient mice, a direct comparison of NM-404 with its precursor, NM-324, both in tumor uptake and systemic excretion of NM-404 compared to NM-324 The dramatic improvement of was revealed. Based on imaging and tissue distribution studies in several rodent tumor models, the C18 analog NM-404 was chosen for follow-up evaluation in human lung cancer patients. The results of preliminary studies are very strongly expected that selective uptake and retention of the agent in the tumor will occur with rapid clearance of background radioactivity from normal tissues, particularly abdominal normal tissues. These results strongly suggest that NM-404 is a valid basis for extension to human studies involving other cancers, especially when NM-404 is radiolabeled with iodine-124, a new commercially available positron emitting isotope. Suggest. The relatively long physical half-life of 4 days obtained with this isotope appears to be well suited to the pharmacodynamic characteristics of NM-404.

Chemical synthesis. In the course of the synthesis of iodinated phospholipid ether analogs, the inventors sought to develop a general synthetic scheme that can vary the chain length of the target compound. The synthetic approach was based on a copper-catalyzed cross-coupling reaction of Grignard reagents with alkyl tosylates or halides ¹⁵ (Schemes 1, 2). The choice of building block for alkyl chain extension was determined by the commercial availability of the starting material.

Scheme 1: Reagents and conditions: (a) Me ₃ SiBr, CH ₂ Cl ₂ ; (b) BrMg (CH ₂ ) ₁₁ OTHP, Li ₂ CuCl ₄ (catalyst), THF, −78 ° C. to 20 ° C .; (c) PPTS, EtOH, 40 ° C .; (d) TsCl, DMAP, CH ₂ Cl ₂ ; (e) BrMg (CH ₂ ) ₃ OTHP, Li ₂ CuCl ₄ (catalyst), THF, −78 ° C. to 20 ° C .; (g) _{BrMg (CH 2) 6 OTHP,} Li 2 CuCl 4 ( catalyst), THF, -78 ℃ ~20 ℃ .

Scheme 1

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, in the presence of 0.5～0.7Mol% of _Li 2 CuCl _4, and further coupled with Grignard reagent derived from 11-bromo undecanol 13 protected with THP. 12- (p-iodophenyl) dodecanol 17 obtained after deprotection of the first coupling product 16 was used for the synthesis of C12 iodinated phospholipids as described above. ^{12, 13} alcohol 17 also served as the starting material for the synthesis of ω-iodophenylalkanols with long chains. For example, coupling of tosylate 18 with Grignard reagent made from bromides 14 and 15 following THP deprotection provides C15 (20) and C18 (22) alcohols, respectively. These alcohols were converted to the corresponding alkyl phosphocholine 5 (NM-397) and 6 (NM-404) by published methods. ^{12, 13} Using a series of reactions previously reported by the inventors, propanediol phospholipid ethers 7 (NM-413) and 8 (NM-412) were synthesized from 3-benzyloxypropanol 25 and 2 -O-methyl-rac-glycerol phospholipid ethers 9 (NM-414) and 10 (NM-410) were obtained from 1-O-benzyl-2-O-methyl-rac-glycerol 26. Radiolabeling with iodine-125 was performed by the isotope conversion method commonly used in the laboratory.

Scheme 2

  biology. The avidity of PLE analogs localized to the tumor was evaluated in several animal models. The PC-3 model directly compares the ratio of NM-404 and NM-412 targets (tumors) to non-targets to select candidates for early human pharmacokinetic studies in prostate cancer patients. The human tumor cell line used to determine The MatLyLu (Dunning R3327 rat) model, a rat prostate tumor line, was placed in control before the rat was placed in control for dosimetry and the tumor-bearing animals were in control to determine the tumor / background ratio. Nine specific analogs were specifically used to screen before being done. Finally, the Walker-256 carcinosarcoma model was used for the purpose of quantifying tissue distribution.

In order to expedite the screening process and to minimize the number of tumor-bearing animals used in tissue distribution studies at multiple time points, a new radioiodinated homologue was developed in rat Dunning R3337 (MAT LyLu strain) prostate. Angiography was performed by gamma camera scintigraphy in a cancer model. Therefore, male Copenhagen rats were injected subcutaneously with MAT LyLu cells (1 × 10 ⁶ cells) in the thigh and 10 to 14 days later a radioiodinated PLE analog in 2% Tween-20 solution. Injections (30-40 μCi) were made. Gamma camera images were obtained at a number of time points including 24 and 48 hours post injection. Homologs (NM-410, NM-413 and NM-414) showing high uptake in the liver, significant abdominal accumulation and retention, or low uptake and residence in the tumor were not subjected to subsequent biodistribution analysis . The tissue distribution of radioactivity in rats bearing Walker-256 carcinosarcoma was evaluated at various time intervals after intravenous administration of a radioiodinated chain length homolog. The first group of compounds tested included three alkyl phosphocholines: shorter chain analog 3 with 7 carbons (NM-396) and two analogs 5 with longer chains (NM -397, C15 alkyl chain length) and 6 (NM-404, C18 alkyl chain length).

  Initial biodistribution experiments were performed with 3 (NM-396, C7 analog), which showed rapid tissue clearance with significant in vivo deiodination. At 24 hours, the amount of radioactivity in the thyroid was 213% ID / g, while the level of radioactivity in all organs examined was <0.10% ID / g. With these studies, it was clear that reducing the number of methylene groups to 7 gives a more hydrophilic molecule that is rapidly excreted by the kidneys. In contrast to compound 4 (NM-346, C12 analog), C7 analog 3 rapidly disappeared from the rat and was not localized to tumor tissue at any time point investigated.

  This observation directed the subsequent effort to evaluate the effect of increased aliphatic chain length on tumor uptake and retention. The tissue distribution of C15 homolog 5 (NM-397) was evaluated using the same Walker 256 rat tumor model. Most normal tissues showed the highest levels of radioactivity 6 hours after dosing, whereas tumor radioactivity increased with time and peaked 48 hours after dosing (ID / g 1.65). ± 0.23%). With the exception of the thyroid, the tumor was at a higher radioactivity concentration than any other tissue examined at 24, 48 and 120 hours. The more rapid excretion of radioactivity in normal tissues compared to tumors is probably due to metabolism and excretion by normal tissues. The accumulation of radioactivity in the thyroid increased throughout the study. This suggested that there was a low level of in vivo deiodination. The radioactivity level in the duodenum was very similar to the tumor level with the maximum level seen 48 hours after administration (1.38 ± 0.24% ID / g).

  In this model, limited results were obtained with C12 analog 4 (NM-346). However, the results are similar to those seen with C12 analog 4, except that the tissue distribution analysis of C15 analog 5 (NM-397) increased twice as much as tumor uptake at 24 hours. Suggested similarities. Retaining uptake and clearance in other organs and tissues was similar between the two compounds.

  The effect of further extending the aliphatic chain to C18 analog 6 (NM-404) indicated the kinetic analysis of this compound. The level of radioactivity peaked in the tumor 48 hours after administration (1.14 ± 0.01% ID / g), although at a slightly lower level, this effect is similar to that of C15 analog 5 (NM-397). It is the same. The amount of radioactivity detected in the liver, kidney and duodenum was significantly lower after administration of C18 compound 6 compared to the same organ in the C15 analog 5 study. Furthermore, C18 analog 6 remained in the blood circulation for much longer than the other chain length homologs investigated. For example, at 120 hours, the blood level of 6 is 0.6 ± 0.1% ID compared to a level of 0.07 ± 0.00% ID / g for the C15 (5) analog. / G. When taking into account very small mass glands, total radioactivity levels in the thyroid were relatively low in both 5 and 6.

To test the transport properties of the PLE analogs, plasma was isolated from Walker-256 tumor bearing rats 7 days after administration of 6 labeled with iodine-125. The distribution of radioactivity in the plasma components of rats inoculated with 6 (NM-404) was investigated. PAGE analysis revealed that most (88%) of the circulating radioactivity after administration of C18 analog 6 was related to the albumin fraction. This finding study the binding of ET-18-OCH ₃ and serum protein is a phospholipid ether prototype, that the majority of the ether lipid (71%) bound to albumin, about 6% is bound to HDL Similar to the results reported by Eibl who found

Comparative contrast studies. Gamma camera scintigraphy imaging shown in FIG. 19 shows that second generation analog ¹²⁵ I-NM-404 after administration 1, 2 and 4 days in immunodeficient SCID mice bearing human PC-3 prostate tumor xenografts. Direct comparison of tumor uptake and body clearance of the shorter generation first generation precursor ¹²⁵ I-NM-324 (2) versus (6). A quantitative scintigraphic comparison of these two PLE analogs demonstrated significant differences in tumor uptake and systemic clearance. The longer chain drug NM-404, which shows rapid tumor uptake and increased retention, is accompanied by rapid excretion of radioactivity throughout the body. On the other hand, tumor uptake and body clearance are substantially delayed with NM-324 even after 4 days of administration. Significant tumor uptake and retention of the C18 analog NM-404 with rapid systemic excretion is clearly defined by the superior imaging properties of NM-404 in this model.

  Broad quantitative tissue distribution results were obtained 1, 3, 5 and 8 days after administration of radioiodinated NM-404 in this model showing rapid excretion of radioactivity from all normal tissues during the 8-day evaluation period. . However, tumor uptake continued to increase until day 5 when the 18% injected dose per gram of tumor was reached. The ratio of tumor to background tissue gradually increased during the experiment, along with successful excretion from normal tissue, due to increased residence in the tumor. The ratio of tumor to background tissue exceeded 4, 6.8, 23 and 9 in blood, liver, muscle and prostate, respectively, 3 days after injection and continued to increase for 5 and 8 days. In addition, when very small mass organs are considered and the data is expressed as a percentage based on the dose administered per organ reference, thyroid levels can range from 26% to 54% of injected dose per gram of tissue. Although in range, these levels are actually very low, indicating a very small percentage of injection dosage.

Kotting et al. Investigated the effect of alkyl chain length on the biodistribution of three alkylphosphocholine (APC) analogs. Kotting's study shows that 1) the compound is administered orally at a concentration that appears to be cytotoxic to the tumor, and 2) the C22 compound contains a double bond in the alkyl chain. It was different. Therefore, due to large dose differences and the unknown bioavailability of oral agents, the study cannot be directly compared with the studies described herein. In the Kotting study, C16, C18 and C22 analogs were orally administered to rats bearing methylnitrosourea-induced breast cancer at a daily dose of 50-120 ∝mol / kg. As the alkyl chain length increased, the levels observed for compounds in the kidney, liver and lung decreased. In contrast to the tracer results obtained with radioiodinated PLE analogs, Kotting and co-authors found that tumor levels increase with increasing chain length, while blood APC levels increase with increasing chain length. Found to decrease. It is expected that ¹⁹ oral administration will result in a substantial amount of degradation of ether lipids in the G1 pathway prior to absorption.

  Tumors were readily visualized by gamma camera scintigraphy using C12 (4), C15 (5) and C18 (6) alkylphosphocholine homologs at both 24 and 48 hours post injection. Rat imaging results are obtained with C15 (7, NM-413) and C18 (8, NM-412) propanediol analogs. On the other hand, uptake in the indicated tumors is accompanied by high levels of radioactivity in the liver and abdomen. Imaging results in the MAT LyLu prostate model were obtained with C15 (9, NM-414) and C18 (10, NM-410) 2 -O-Me glycerol analogs, with little drug uptake in the tumor Or showed high radioactivity levels in the liver and abdomen with no uptake. Differences in clearance and amount of radioactivity from non-target tissues including the liver and intestinal tract have significant effects when applying radioiodinated phospholipid ether analogs as human contrast agents. Uptake with non-target tissue can reduce the effect of radiodiagnosis imaging by creating high background activity against injected radioactivity or by overexposing the radiosensitive tissue. Preliminary clinical trials with 2 (NM-324, NM-346 metaiodo nuclear isomers) in cancer patients give superior tumor uptake while accumulation in non-target tissues including liver, kidney and bladder Limited by radiation dose measurements.

  The strategy was to investigate the alkyl portion of the phospholipid ether analog structure and determine its role in tumor localization and retention. Qualitative rat whole body screening scans obtained in MAT-LyLu tumor-bearing rats with longer chain length radioiodinated PLE analogs reveal sufficient tumor uptake to allow detection became. However, in follow-up tissue distribution studies, the amount of radioactivity detected in non-target organs rapidly decreased with increasing chain length from C12 to C15, C18. This sufficient decrease in the amount of radioactivity in non-target tissues was accompanied by a relatively small decrease in the level of radioactivity present in the tumor. Furthermore, C18 analog 6 (NM-404) tended to retain a much longer ring shape than the C12 (4) and C15 (5) analogs. A longer plasma half-life can be expected to provide further opportunities for uptake of C18 compound 6 in the tumor as the compound circulates continuously through the vasculature. This extended plasma half-life may be the result of strong binding between the probe and albumin. The uptake and transport of radiolabeled PLE by plasma components may also be an important factor related to the retention of these compounds in tumors. Indeed, increasing the chain length from C7 to C18 results in an increase in the lipophilicity of the PLE analog. Greater lipophilicity can increase the affinity of these compounds for the cell membrane and can alter binding to plasma components. Uptake and transport in the blood circulation by endogenous lipoproteins (eg, LDL and HDL) can also affect biological distribution in tumors.

In preparation for human clinical trials, unlabeled NM404 was subjected to an independent (University of Buffalo Toxicology Research) acute toxicity assessment at 1200 times the expected contrast dose in rats and rabbits. The drug was well tolerated and acute toxicity was found at this dose level. NM-404 is a non-small cell lung cancer (NSCLC) patient because of its selective tumor uptake and retention characteristics in various rodent tumor models and subsequent excellent safety characteristics in rats and rabbits. We chose to do our first human pharmacokinetic assessment. The patient received planar gamma camera scintigraphy after receiving an injection of ¹³¹ I-NM-404 (<1 mCi). Preliminary human results (n = 3) demonstrated tumor uptake and enhanced retention in primary lung tumors (FIG. 25). However, with its precursor NM-324, high uptake values were observed in the liver, whereas the radiation levels in the liver and abdomen were much lower than those in NM-404. This suggests that the drug may be evaluated in other abdominal tumors, including those related to the colon, prostate and pancreas.

  Conclusion

  In summary, the rationale behind the design of the novel iodinated PLE analogs described in this document is that the chain length in the hydrophobic portion of the phospholipid molecule and other factors for tumor uptake and retention It was used in efforts to evaluate the effects of structural modifications. Decreasing the chain length from C12 to C7 resulted in little or no accumulation of the compound in the tumor and rapid clearance up to 24 hours after administration in tumor bearing rats. In tumor-bearing rats, increasing chain length produced the opposite effect, and C15 and C18 analogs delayed plasma clearance and promoted tumor uptake and retention. Tumor uptake demonstrated by propanediol analogs 7 and 8 was accompanied by slightly higher radioactivity levels in the liver and abdomen 24 hours after injection into tumor bearing rats. The addition of 2-O-methyl moieties at 9 and 10 significantly delayed tumor uptake. In a human PC-3 bearing immunodeficient mouse, by comparing directly between NM-404 and its precursor NM-324, both in tumor uptake and systemic excretion of NM-404, Compared to NM-324, a dramatic enhancement was revealed. NM-404 had excellent contrast properties over other analogs tested in several animal models. Thus, further evaluation of this second generation PLE analog in human lung cancer patients was warranted. Preclinical results in humans have shown desired tumor uptake and retention characteristics similar to those previously seen in animal models. However, in contrast to NM-324, which is a shorter chain precursor, NM-404 showed liver, kidney and abdomen with significantly lower background radioactivity levels. This suggests that in addition to providing a perspective on lung tumor imaging, further evaluation of this agent in human colorectal, pancreatic and prostate cancer patients is warranted. Furthermore, the lack of radioactivity in the bladder suggests that there is little renal clearance of the drug or its metabolites over the time point investigated. This represents a significant advantage over 18F-fluorodeoxyglucose (FDG), a PET agent that is routinely used for tumor imaging today. FDG is prominently excreted in the kidney, thus preventing contrast in the prostate region.

Since PLE analog tumor targeting strategies are thought to affect selective tumor retention over time, relatively short half-life nuclides (eg, even ¹⁸ F or even ^99m Tc) are not Not practical for labeling -404. Radiolabeling NM-404 with iodine-124, a relatively new positron emitting isotope with a half-life of 4.2 days, as ¹³¹ I-NM-404 has been successfully piloted in current lung cancer imaging studies It is now essential to evaluate tumor detection efficiency by PET and PET. It has been reported that ¹²⁴ I PET imaging is more than 40 times the sensitivity of planar ¹³¹ I-gamma imaging, PET. Unlike conventional gamma camera imaging, PET also provides significant resolution enhancement and three-dimensional imaging capabilities, as well as superior quantitative properties compared to planar scintigraphic imaging. The 4 day long physical half-life of this PET isotope is well suited to NM-404 uptake and retention kinetics in tumors, and we are in the process of expanding imaging studies using NM-404 with PET. I'm on the way.

Non-small cell lung cancer (NSCLC)

  Phospholipid ether analog (PLE) molecules have unique biochemical and pharmacological properties that result in a high degree of tumor selectivity. Unlike FDG, which accumulates nonspecifically in both malignant and non-malignant hypermetabolic tissues, radioiodinated PLE analogs are at high levels in a variety of mouse and human tumors. The present inventors have shown that they are selectively retained and do not accumulate in normal or inflamed tissues. The phospholipid ether family (PLE) is characterized by the presence of a long chain alkyl or alkenyl alcohol linked to an ether linked to a glycerophosphocholine molecule commonly found in mammalian cells as a minor component of the total phospholipid content. It is done. There are many subtypes among PLE, and one of the most widely studied subtypes is structurally simple alkylphosphocholine (APC). I-125-NM404 is part of this APC family.

The present invention teaches a methodology for preliminary human PET imaging data involving the use of a second generation PLE analog, NM-404, in imaging patients suffering from NSCLC. In the preclinical model, we have selectively retained NM-404 in (a) 27/27 tumor models, including lung and brain tumors and PC-3 prostate bone metastases, and (b) normal It was shown that it was not retained in tissue, pre-malignant tissue or hyperplastic tissue and (c) not retained in inflamed tissue. In the first human pharmacokinetic study using ¹³¹ I-NM404 in a patient suffering from NSCLC, we have found that ¹³¹ I-NM404 is safe and optical scintigraphy to detect tumors. It was found that the time point of graphy imaging was 24-48 hours. These studies also revealed significantly lower liver levels and background activity levels for the initial expected analogs and confirmed that the drug did not cross the intact blood brain barrier.

Although sufficient for early human pharmacokinetic studies, the lack of contrast and plane contrast properties for iodine-131 scintigraphy further develops NM404 for PET imaging It affects. We have recently radioiodinated NM404 in excellent yield using commercially available iodine-124, a relatively new and long half-life PET isotope. Its half-life appears to be ideally matched with the pharmacokinetic analysis results of NM404. Primitive microPET scans were obtained with ¹²⁴ I-NM404 in xenografts, confirming that spontaneous mouse and rat tumor models have universal tumor avidity and increased retention. Extending these results to PET scans is now necessary to accurately characterize and quantify the distribution characteristics of this drug in vivo. The main purpose of this proposal is to further develop NM404 for PET / CT imaging in NSCLC patients using this radioisotope.

The present invention provides: (a) the effect of imaging primary NSCLS tumors using ¹²⁴ I-NM404 PET / CT in patients with NSCLC who have undergone resection by comparing preoperative images with pathological findings (B) Specific tumor accumulation and metabolic fate of NM404 in NSCLC patients who have undergone resection, and correlation between tumor retention and decreased phospholipase D activity; (c) these results are expressed as FDG PET / Preliminary study data on sensitivity to image local and metastatic tumors using ¹²⁴ I-NM404 PET / CT in patients with NSCLC by comparison with CT scan; and (d) with isolated lung nodules Diagnose the patient or lung sarcoidosis or granulomatous infection (eg fungal or bacterial infection, or bacterial pneumonia) By contrast the ^patients, to study the preliminary test data relates to the sensitivity of imaging with 124 I-NM404 PET / CT.

Due to its increased retention in tumors, NM404 labeled with ¹²⁵ I has recently undergone a significant tumor regression in A549 human lung tumor xenograft-bearing SCID mice (see below). NM404 has been developed as a diagnostic and therapeutic agent that has the potential to be authentic and universal because it exhibits both diagnostic and medicinal utility.

NM404 is currently being evaluated in 27 animal tumor models including several lungs, and it is clear that once the drug enters the tumor cells, it reaches the end of metabolism and is trapped. It is. Increased tumor retention of this drug is demonstrated in human adrenal tumor xenografts transplanted into SCID mice (FIG. 14). NM404 also resides in spontaneous mouse lung tumors (Figure 13). Using NM404 labeled with ¹²⁵ I, we were able to image breast and prostate tumors in mice over 60 days. The retention characteristics in the augmented tumor can significantly enhance the radiotherapeutic effect of the drug. Contrast and tissue distribution studies are performed in a mouse model aimed at determining uptake characteristics in a wide range of tumor models and are summarized in Table 2.

Rationale for isotope selection in clinical research

  We investigated NM404 labeled with iodine-131 in a preliminary pharmacokinetic study, but this is not optimal for imaging. This is because iodine-131 scintigraphy produces images with limited resolution and little anatomical detail. However, iodine-124, a novel long half-life isotope, produces tomographic PET images that are displayed with corresponding CT images, thereby providing significantly better anatomical and functional details.

Because the PLE analog tumor targeting strategy appears to selectively stay in the tumor over time, relatively short half-life nuclides (eg, even ¹⁸ F or even ^99m Tc) are currently practical to label NM404. Not right. Although the use of other isotopes, including iodine-123, may ultimately prove suitable for use of NM404 in certain tumors, the current focus is on a combined PET-CT scanner The recent success of tumor imaging will develop this agent's PET imaging capabilities. The unsurpassed diagnostic accuracy provided by biochemical or functional tumor contrast PET agents combined with the precise anatomical accuracy provided by CT is now the gold standard for tumor imaging. However, labeling PLE analogs with iodine-124, a relatively new PET isotope, is very advantageous and the physical half-life (4 days) is good for tumor uptake and retention kinetics of PLE. Fit. Labeling NM404 with iodine-124 represents a natural extension of recent research using gamma-ray radionuclides based on the physical half-life of 4 days. ^It has been shown that PET imaging with ¹²⁴ I provides a sensitivity that is over 40 times that of planar ¹³¹ I-gun mammography. PET, unlike conventional gamma camera imaging, not only shows significant resolution enhancement and 3D capability, but also used in combination with combined PET-CT by incorporating the advantages of attenuated correction provided by CT Provides excellent contrast evaluation methods when done. The successful detection of ¹³¹ I-NM404 in a recent lung cancer imaging study evaluates the tumor detection effect of NM404 labeled with iodine-124 by PET to overcome the limitations inherently associated with planar scintigraphy Doing is now essential.

Successful radiolabelling of NM404 with iodine-124

  We obtained high specific activity of sodium-iodine-124 in 0.1N NaOH from Eastern Isotope (Sterling, VA). The NM404 radiolabel achieves an isolated radiochemical yield of greater than 60% by modification of the isotope conversion method. Briefly, a 2 ml glass vial is filled with 10 mg of ammonium sulfide dissolved in 50 μl of deionized water. Glass beads were added, a Teflon treated diaphragm and screw cap were added, and the vial was swirled gently. In less than 30 μl of 0.01N aqueous sodium hydroxide, add 10 μg of a solution of NM404 stock (in 10 μl of ethanol) followed by aqueous sodium iodide (I-124, 1-5 mCi). Gently swirl the reaction vial. A 5 ml disposable syringe containing glass wool in series is attached at the end of the needle to another 5 ml syringe filled with a small chunk of charcoal. The glass wool syringe serves as a concentration chamber for trapping evaporated solvent, and the charcoal syringe serves as a concentration chamber for trapping free iodide / iodine. The reaction vessel is heated at 150 ° C. for 45 minutes using a heat block apparatus. Thereafter, four 20 ml volumes of air are injected into a reaction vial connected to a 25 ml disposable syringe and released through a double trap attachment. The temperature is raised to 160 ° C. and the reaction vial is then heated for 30 minutes. After cooling to room temperature, ethanol (200 μl) was added and the vial was rotated. The ethanol solution is passed through a pre-equilibrated Amberlite IRA 400-OH resin column to remove unreacted iodide. The eluent volume was reduced to 50 μl via a nitrogen stream (using a charcoal syringe trap) and the remaining volume was injected into a silica gel column for purification (Perkin Elmer, hexane / isopropanol / water (52:40). : 8) 3 μm × 3 cm disposable cartridge column eluting at 1 ml / min). Final purity is determined by TLC (silica gel-60 supported on plastic eluted with chloroform-methanol-water (65: 35: 4, Rf = 0.1)). The HPLC solvent was removed by rotary evaporation and the resulting radioiodinated NM404 was solubilized with 2% aqueous polysorbate-20 and passed through a 0.22 μm filter in a sterile vial. Radiochemical purity is typically greater than 99%.

¹²⁴ In vivo PET imaging of mouse tumors with I-NM404

Brain tumor. We evaluated the imaging properties of NM404 in C6 glioma (3-5 mm diameter) tumor bearing rats and sham-operated rats. Tissue distribution analysis was performed with ¹²⁵ I-NM404 24 and 48 hours after injection, and separate groups of animals were microPET scanned at various time points after injection of ¹²⁴ I-NM404. In normal brain tissue, biodistribution analysis showed minimal NM404 radioactivity. However, at 24 and 48 hours, the tumor / brain ratio (% injected dose / g) was 10.6 and 12.0, respectively. (FIG. 15) Tumor uptake of NM404 was supported by histology. These preliminary experimental results obtained in the rat glioma model suggest that NM404 is quite promising for detecting malignant primary and metastatic brain tumors.

Lung, prostate and pancreatic tumor models

Results of preliminary microPET imaging with ¹²⁴ I-NM404 in lung, prostate and pancreas mouse models are shown in FIGS. A common feature of all microPET imaging obtained in animal models is the lack of bladder activity at any time point. Human pharmacokinetic studies supported this finding that only 4% of the drug was cleared in the kidney within 4 days after intravenous injection (mostly excreted by the G1 pathway). In all cases, NM404 showed significant tumor avidity regardless of its location in the body. Although tumors (especially abdominal tumors) generally increase significantly over time, tumor uptake typically occurs within 6 hours of injection (scene). ).

Stimulated by these and other observations, we have recently conducted a pilot therapeutic study of small-scale ¹²⁵ I-NM404 in SCID mice bearing human A549 lung tumor xenografts. ¹²⁵ I-NM404 was administered to a group of 6 mice either at a single dose or alternatively at 3 doses (once a week for 3 weeks), and the divided groups were not used as comparative examples. An equal dose of labeled NM404 was received. The single dose was 50 or 500 μCi and the repeated dose group received a 50 μCi dose for a total of 3 weeks. Tumor growth was monitored for 10 weeks after the final injection. Preliminary experimental results show significant regression in high dose tumor growth and probably similar response in the three dose group. However, these arms of the study are still ongoing. New battles are currently being started to cover dose levels between current ones in order to more accurately assess the pharmaceutical potential of this drug. Even at the 500 μCi dose, the mice did not show any signs of toxicity.

Clinical trial

  In a Phase 1 NSCLC study at the University of Wisconsin, NM404 was administered at a dose of tracer (0.3 μg / kg body weight) for imaging. A 70 kg subject would therefore have received approximately 21 μg of NM404. However, recent improvements in conversion labeling procedures result in much lower doses being injected. Recent improvements in labeling and specific activity allow us to reach doses in the range of a total dose of 142.9 ng / kg BW corresponding to 0.22 nmol / kg BW or 0.010 mg per 70 kg patient Met. Further improvements are expected to be based on the following novel labeling methodology. The improvement will reduce the amount of compound needed by less than 1/50. In view of their future improvements, the clinical dose of I-125-NM404 that we intended to be given in the clinical trials conducted is approximately 3-5 ng / kg BW or about 70 kg per patient. 250 ng.

  Some of the phospholipid ethers known as alkyl phosphocholines have a wide range of pharmacological activities and have been extensively studied as anti-cancer and anti-leshmanial agents in animal models and humans at micromolar concentrations. Although disruption of membrane lipid metabolism is seen in tumor cell membranes, not only the mode of uptake but also the exact mechanism (s) of action are clearly undefined. Miltefosine, hexadecylphosphocholine, has been reported to have an LD50 of 606 nmol / kg in rats that achieves a maximum tolerated dose of 39 nmol / kg over a 4-week period. In clinical trials in humans, a daily oral dose of 150 mg (3 × 50 mg) was well tolerated with minimal side effects (nausea and vomiting) (Planting AS, Stutter G, Verweij J. European Journal of Cancer 1993; 29A : 518-519).

Toxicity studies conducted at NM404

  This section summarizes four formal GLP toxicity studies conducted with NM404. These studies are:

  Formal toxicity studies of NM404 in male rats and rabbits were conducted at the Toxicology Research Center at State University of New York in Buffalo under the supervision of Dr. Paul Kostyniak. Drug vehicles and drug products were provided by Dr. Raymond Counsell at the University of Michigan to Dr. Kostynia for the studies described in the attached Study 27 and Study 28 overview. Since no significant toxic effects were noted at the 4 mg / kg dose (this amount is approximately 200 times the expected contrast dose at that time), we have performed under this invention The expected therapeutic dose for a given clinical trial was estimated to be approximately 2860 times. Human safety studies of unlabeled NM404 began in normal human men with an amount 10 times the expected contrast dose and about 21 times the expected therapeutic dose. The results again showed no toxicity due to the drug substance. This toxicity study was performed under GLP conditions.

  Subsequently, researchers at the University of Wisconsin conducted a toxicity study of unlabeled NM404 at the Toxicology Research Center in SUNY-Buffalo to expand the patient population studied in the NSCLC Phase 1 clinical trial. (Study 31 and Study 32). No significant toxic effects were noted at the 0.04 mg / kg dose (which is approximately 200 times the revised contrast dose at that time). Therefore, we estimated that the expected therapeutic dose for clinical trials was approximately 286 times. A human safety study of unlabeled NM404 was initiated in normal human women with an amount of 10 times the initially expected contrast dose and about 21 times the expected therapeutic dose. Furthermore, no significant toxic effects were noted in either female rats or rabbits. This toxicity study was performed under GLP conditions.

  Toxicity studies were then designed to reach up to 200 times the expected dose in human studies. Thereafter, improvements in conversion labeling methodology (see method 2 in CMC section) will likely result in a reduction of less than 1/50 in the amount of compound required for the reaction. Therefore, recalculation of the toxic dose showed that there was clearly no toxicity at doses of at least 10,000 x expected clinical dose. During normal phase 1 safety, pharmacokinetic and dosimetric studies in normal volunteers and NSCLC patients, normal male (UM1) or female (UW) volunteers, or any NSCLC who participated in the study No toxicity was observed in any of the patients (UW).

  Control and test products:

  The test product was a solution of C-NM404 (active ingredient) containing an inert ingredient (vehicle). The control solution for this study was a vehicle without active ingredients.

The test product was formulated as follows:
(1) Active ingredient: 2 mg / mL C-NM404
(2) Inactive ingredients: 2% polysorbate 20 in sterile water (injection grade)

The control was formulated as follows:
(1) Inactive ingredients: 2% polysorbate 20 in sterile water (injection grade)

  This control product and test product was published on October 29, 1998 by Professor Raymond E. I received it from Dr. Counsell. Four vials of test product labeled “2% polysorbate 20 / NM-404 (2 mg / ml), MAL-V1-82, 2% polysorbate 20 / sterilized in water” at the Toxicology Research Center at the University of Buffalo, the study site of this study. , And four vials of the control product labeled “Control Vehicle—2% Polysorbate 20 in Sterile Water, MAL-V1-83” were listed and stored at room temperature.

  Administration: The test product (C-NM-404) was administered at a dose greater than 200 times the expected clinical dose. Control and test rats were injected intravenously into the veins on the side of the tail. Rats were intravenously injected with test or control products at 2 ml / kg body weight using a 25 caliber needle and a 1 ml syringe. Injection was performed by alternating between rats from the control group and rats from the test group. Injections were carefully made at time intervals longer than 30 seconds up to 1 minute. The injection to control rat # 27-01 was at 9:03 am and the last injection to test rat # 27-16 was at 11:01 am. The following rats were moving during the injection and received multiple injections: Control # 6 (2 places), Test # 9 (3 places), Test # 15 (3 places).

  In human application, ET-18-OCH3, an authentic PLE, and therefore less similar to NM404 than miltefosine, but (edelfosine, mouse LD50 (oral) 200 mg / kg) is intravenously 5% HSA It is administered at a dose of 15-20 mg / kg / d at a medium 5 mg / ml. The maximum tolerated dose is 50 mg / kg. Side effects reported for this agent include liver function and hemolysis impaired by pulmonary edema by 4 hours after injection. (Berdel WE, Fink U, Rasetter J. Lipids 1987; 22: 967-969). Considering that the total dose of NM404 is less than 1/10000 of the daily dose of miltefosine, no toxic event is expected.

  Study Procedure: Rats were observed by LAF personnel for signs of acute toxicity from the time of injection to 3:30 pm. The rats were weighed 5 times a week (Monday to Friday) and recorded in kilograms. On December 17, 1998, rats were anesthetized with pentobarbital sodium (65 mg / ml, Lot # 970789, expiration date: February 1, 2000) administered intraperitoneally. A cardiac puncture was then performed using a 20 caliber needle and a 10 ml syringe to collect blood samples with hematological CBC and clinical blood chemistry. Rats were exsanguinated and killed. Thymus, heart, lung, spleen, kidney, liver, brain and testicles were collected, macroscopically examined, weighed, and dissected for pathology. Tissue samples (excluding the thymus) were placed in jars of fixative “Z-fix” for histology.

  Sixteen Sprague-Dawley rats were received from Harlan-Sprague Dawley, Indianapolis, Indiana. All rats were males born on the same day and appeared healthy. These rats were bred on Laboratory Animal Facility and optionally fed food and water. Each rat was given a two part number (indicative) starting with 27 (study number) followed by a “unique” number from “01” to “16”. A hole was drilled in the right ear of each rat based on a unique number from “01” to “16”. Control rats were numbered from # 27-01 to # 27-08, and test rats were numbered from # 27-09 to # 27-16. A unique number was also assigned to each cage to indicate which rats were housed in that cage. There were four control rat cages and four test rat cages. Rats were weighed. The average weight of the control group was 0.234 kilograms and the test group was 0.238 kilograms. Two groups of 8 rats were constructed by random assignment. Rats were weighed daily until the end of the 14th day study. Test product, dose and mode of administration, batch number: C-NM-404 (MAL-V1-82) 2 mg / ml, administered via tail vein injection over a 30-60 second treatment period: single Single dose comparative treatment, dose and mode of administration, batch number: 2% polysorbate 20 in sterile water, 2 ml / kg body weight, administered via tail vein injection for longer than 30-60 seconds.

  Safety: On day 14, both groups of blood samples are analyzed hematologic and clinical chemistry. In addition, the following organs are excluded for pathological reports and histological slides: brain, lung, liver, heart, kidney, spleen and testis. For each rat, an organ weight and organ / body ratio compilation chart can be compiled.

  Statistical methods:

  Differences between groups in body weight and biochemical parameters are compared using a t-test.

  Safety results:

  None of the rats showed abnormal behavior during this period or throughout this 14-day study. The tail vein injection site was checked daily when rats were weighed and no tissue side effects were noted in any rat. The average body weight of the rats in the control and test groups was not significantly different except that the body weight was not significant in both groups and periodically decreased daily.

  At Buffalo SUNY, a pathologist performed a macroscopic examination of the tissue one week after sacrifice. No visible lesions were noted in the test or control group. The incised surface of the tissue was then analyzed by examination with an optical microscope. There were no changes in histopathology in the organs examined that could be attributed to the administration of the test substance. Rat # 27-12 inoculated with the test substance has a small focal area of myocardial injury (early stage infarction). This change is not seen in other compartments that were found to have progressed as well as interpreted as very small. The pathologist felt that it was due to some unexplained changes, as no lesions were seen in other animals inoculated with the substance. Since the lung does not show histopathological changes, it is not due to an infection caused by the lung.

  Clinical blood chemistry and hematology results were investigated for any obvious value (which was greater or less than the reference range). These numbers were to compare groups using a t-test with a p-value of 0.05. A t-test was performed on: phosphorus, sodium, potassium, AST, ALT, alkaline phosphatase, globulin, A / G ratio, glucose, WBC, RBC and hemoglobin. There was no significant difference.

  Conclusion: No acute toxic effects were observed due to the test product. There was no significant difference between the test group and the control group.

  Similar tests were performed on male and female rabbits and female rats. No side effects caused by the test product were noted.

Preclinical pharmacology:

  The inventors working on the development of safe and effective cancer therapies design small molecule carrier molecules. The molecule has the ability to selectively stay in cancer tissue, but not to stay in tissue that is not cancer or minimally. Expansion of this approach to radiotherapy develops selective delivery of radiopharmaceuticals that minimizes radiation-induced damage to normal tissues while depositing therapeutic levels of radiation within the tumor mass . This technology allows for the inherent biochemical and pharmacological properties of phospholipid ethers (PLE's) and in particular their subgroups of alkylphosphocholine analogues (eg NM404, which exhibits a high degree of tumor selectivity). Based. Phospholipids are an integral component of the cell membrane, provide structural integrity, and are closely associated with a variety of cell signaling processes. Phosphatidylcholine is well known as lecithin and is an example of such. Phospholipid ethers, on the other hand, represent a small number of subclasses of phospholipids that are also present in membranes. As the name suggests, these lipids contain ether linkages rather than ester linkages at the C1 site. Platelet activating factor (PAF) represents one of the more well-known phospholipid ethers.

  Based on his initial findings that several animal and human tumors contain much higher concentrations of naturally occurring ether lipids in the tumor cell membrane than in normal tissues (Snyder, F And Wood R. Cancer Res. 1968; 28: 972-978, Snyder F. and Wood R.Cancer Res. 1969; 29: 251-258), Snyder is the result of the accumulation of ether lipids in tumors. It was proposed to further reduce the ability of tumor cells to metabolize lipids. The most common hypothesis is that phospholipid ethers are trapped at the tumor membrane because the cell membrane is probably not capable of being metabolized and removed by the phospholipase enzyme. This hypothesis reveals that lipid extracts obtained from tumors after administration of radioiodinated phospholipid ether are only intact agents, while urine and stool analysis reveals only metabolites (Plotzke, KP, et al., J Nucl Biol Med, 1993; 37: 264-272). Therefore, the reason why the tumor stays in PLE is due to the difference in the clearance rate of PLE between normal cells and tumor cells.

  Extensive structure-activity relationship studies have resulted in the synthesis, radiolabeling and evaluation of over 20 phospholipid ether analogs as potential tumor selective contrast agents. Iodinated APC analogs were readily labeled with all iodine radioisotopes using isotope conversion methods. These PLE analogs are specifically designed to incorporate aromatic radioiodine to make the molecule stable against in vivo deiodination. Low levels of thyroid activity in both pre-clinical imaging and tissue distribution studies (both% injection dose / g and% injection dose / organ criteria) have been demonstrated in vivo for radioiodinated PLE analogs. Guarantees the stability of

  In a library of phospholipid ether (PLE) compounds, NM324 [12- (3-iodophenyl) -dodecylphosphocholine] initially showed the most promise in animal tumor localization studies. A variety of tumors, including breast tumors, prostate tumors, squamous cell carcinomas, ovarian tumors, colorectal tumors and melanomas, were successfully visualized by scintigraphy using NM324. Observe unacceptable accumulation in liver tissue and identify PLE compounds with excellent tumor localization and background clearance properties during the first human pharmacokinetic study with NM324, a prototype agent Further experiments were conducted for this purpose. Based on this study, NM404 [18- (4-iodophenyl) -octadecylphosphocholine] enhances its ability to localize in tumors, increases metabolic clearance from the liver, and more in plasma It emerged due to the long half-life. Important observations demonstrated the ability of NM404 to localize in lymph node metastases and were clearly delineated by scintigraphy in a metastatic prostate tumor model with no retention in uninvolved lymph nodes.

The major compound, NM404, has been evaluated to date in over 25 animal tumor models and all tumor models and tumor types as far as studied, and NM404 exhibits tumor selective retention. Increased tumor retention of ¹²⁵ I-NM404 has been demonstrated in mice for a period of 20-60 days after injection. Such very extensive and prolonged tumor retention properties can significantly enhance the radiotherapeutic effect of the agent (especially isotopes with slow decay of radioactivity, such as iodine-125).

Extensive biodistribution data for the prototype drug ¹²⁵ I-NM324 in several tumor models indicates an 8: 1 tumor-to-blood ratio at a later post-injection time. It became clear that In one such example in a rat breast cancer model, the tumor-to-normal tissue ratio reaches its highest at 96 hours, the tumor-to-blood ratio is 8.6, and the tumor-to-muscle ratio is 20: 1. there were. Furthermore, the heterogeneity of the biodistribution of PLE-related radioactivity in tumors was demonstrated by microautoradiography studies. This study showed that PLE radioactivity was present only in viable tumor cells located in the outer region rather than the central necrotic region. Comparative biodistribution data for NM324 and NM404 were obtained in the SCID mouse prostate tumor model and the A549 lung cancer tumor model. These studies revealed a large ratio of tumor to normal tissue and tumor uptake in more than 25% of the injected dose of NM404 in the tumor. This therefore supports the desire to study the biodistribution of NM404 in humans.

Mechanism of action

  Metabolic research

  Formal metabolic studies were performed with several PLE analogs, including NM324, a precursor of NM404. In these studies, each drug was tested to determine its ability to act as an enzyme substrate for PLE metabolism. Three major enzymatic pathways are involved in PLE metabolism. O-alkylglycerol monooxygenase (AGMO) is responsible for cleaving the alkyl ether bond at C1 to form either a long chain fatty alcohol or a later corresponding fatty acid. Phospholipase C (PLC) and phospholipase D (PLD), on the other hand, yield glycerol or phosphatidic acid products, respectively. NM324 was not a substrate for this enzyme when compared to [3H] -lyso-PAF (platelet activating factor), which is extensively metabolized using microsomal AGMO enzyme preparations. In a similar manner, NM324 was analyzed as a substrate for PLC isolated from Bacillus cereus and significantly hydrolyzed to 1-palmitoyl-2- [3H] -palmitoyl-L-3-phosphatidylcholine (DPPC). In comparison, no hydrolysis was performed.

  Finally, several PLE analogs were subjected to phospholipase D (PLD) assay. In addition to phosphatidic acid when the enzymatic reaction is performed in the presence of ethanol, the PLD (which is isolated from cabbage) is that the cabbage form is a product of the phosphatidylethanol type. Was similar to mammalian PLD. Several PLE analogs subjected to these assay conditions produced phosphatidylethanol adducts, indicating that they can interact with PLD. We believe that NM404 is a metabolic substrate for human phospholipase D, and that the relative lack of phospholipase D in the cancer cell membrane underlies a mechanism for tumor selective retention of NM404. However, as is known from the literature (references ???), it is still unclear why cancer is PLD deficient in the membrane.

  Several NM404 precursors were also subjected to in vitro metabolic studies in a variety of cell lines including Walker tumor cells, rat muscle (H9c2) and rat hepatocytes. In these studies, the degree of metabolism was determined based on the radiolabeled product formed after incubation at various times, and the results were normalized to the number of cells or the amount of cellular protein. The lipid extract resulting from the incubation medium and cell suspension demonstrated little production of PLE metabolites in Walker tumor cells, while both myocytes and hepatocytes during the 48 hour study period Has demonstrated the significant production of metabolites. These results correlate well with in vivo biodistribution studies completed for all analogs. Although some studies have been completed, the role of metabolic traps in the uptake and retention of radiolabeled PLE analogs in tumor cells has not been well defined and is still an active area of testing recently. We believe that NM404 can enter the cell membrane of whole cells, but is excreted from non-cancerous cells through rapid metabolism, whereas it is trapped in cancer cells by the lack of appropriate metabolic enzymes. .

  PLD assay

  With the clear universality of NM404 tumor retention in animal tumor models and the first confirmatory results in human lung cancer trials, we began to investigate the mechanism of action of this agent. Although the membrane metabolism of PLE analogs is controlled by a variety of phospholipases, we focused on the initial approach to phospholipase D (PLD) activity. This approach is based on the hypothesis that cellular uptake and retention of NM404 is inversely related to the amount of PLD in the tumor cell membrane relative to normal cells.

  For these findings, preliminary assessments of PLD protein activity and PLD mRNA quantification by RT-PCR assay were performed in several mouse tumor cell lines and compared to normal liver. Such mouse tumor cell lines include mouse tumor cell lines hepa-1 (liver cancer), CT26 (colorectal adenocarcinoma) and TS / A (breast cancer). These experiments revealed that both PLD protein activity and mRNA levels were significantly lower in tumors than normal liver tissue (p <0.05, T test) (Table 1).

  In conclusion, the mechanism of selective retention of NM404 may be due to a decrease in the membrane level of PLD and therefore may interfere with NM404 metabolism and clearance from cells. The idea of performing the initial enzyme substrate assay with cabbage-derived PLD showed that NM404 was indeed an excellent substrate for this enzyme. This supports the findings in uptake and retention studies in cell culture in vitro. The study showed that PLE analogs were captured by normal cells (including normal levels of PLD) and then metabolized. If the malignant tumor cells have normal complement of PLD, the drug is similarly metabolized and excreted from the tumor cells. Conversely, it can be deduced that the lack of metabolism and clearance of the agent from malignant cells supports the hypothesis that these neoplastic cells lack PLD compared to surrounding normal host cells.

Other research

  Mechanistic studies with PLE analogs: NM324 and NM404 are similar in structure to miltefosine (hexadecylphosphocholine), the most widely studied antitumor ether lipid in Europe. The antitumor properties of miltefosine and several other antitumor phospholipid ether analogs have been demonstrated in a wide range of tumor cell lines. Tumor cell lines include prostate tumor, bladder tumor and teratocarcinoma, mouse leukemia and human leukemia, and lung cancer, colon cancer, ovarian cancer, brain cancer and breast cancer (Lohmeyer M, Bittman R. Drugs of the Future 1994). 19: 1021-1037). In contrast to many anticancer drugs, these phospholipid ether analogs do not bind directly to DNA and are not mutagenic. Although the exact antiproliferative mechanism of action has not been determined, phospholipid ether analogs clearly act on several tumor cell sites. These compounds are involved in a variety of cellular effects including transport, promotion of cytokine formation, induction of apoptosis and various major lipid metabolism and cell signaling enzymes (most of which are located in the cell membrane). Interference). Although unclear matters remain regarding the mode of PLE incorporation into cells, most evidence currently supports the idea that these ether lipids are absorbed directly into the cell membrane where they accumulate. A widely accepted idea is that these agents act by disrupting membrane phospholipid metabolism; however, cell distribution studies of these agents have been performed in naturally occurring cellular compartments during homogenization. Limited by redistribution and subcellular fractionation procedures. In contrast to the tracer contrast dose used in the imaging and biodistribution studies quoted by the inventors (several μg), the anti-tumor effect was only seen at doses generally exceeding 150 mg per day (Planting AS, Stoper G, Verweij J. European Journal of Cancer, 1993; 29A: 518-9; Verweij J, Planting A, van der Burg M, Stoper G. Journal & J. Journal & Cancer. Muschiol C, et al. Lipids 1987; 22: 930-934).

Mechanism of action

  The general mechanism of action is that phospholipid ethers (eg NM404) become trapped in the malignant cell membrane due to their inability to be metabolized and excreted. Tumor excision after administration of radioiodinated phospholipid ether showed only the presence of intact drug, but urine and stool analysis showed that only metabolites. Underlying this concept is therefore the different clearance rates of phospholipid ethers of normal cells versus tumor cells. Preliminary results obtained with more than 27 xenografts and spontaneous tumor models have universally shown that NM404 has a selective and increased retention in tumors.

Isotope selection for treatment

  The inventors believe that iodine-125 is the most appropriate radioisotope for use in combination with NM404 targeting the spine. The reason is:

  The long half-life of the iodine-125 isotope is perfectly compatible with NM404, which has a long and stable tumor residence, which delivers a radiation dose that can be treated for an extended period of time.

  The effect of iodine-125 is caused by both low energy gamma / X-ray irradiation and Auger electrons. All of these have very limited treatment distances. Since NM404 is taken up into the tumor, iodine-125 can efficiently deliver a tumor dose, but does not harm the surrounding healthy tissue.

  Iodine-125 has Auger electrons as one of the radiation attenuation byproducts (FIG. 2). Auger electrons produce a significant biological effect, but the treatment distance is very short. Since NM404 is taken directly into the whole cell membrane of cancer cells (including the nuclear membrane), the therapeutic distance to DNA is very low. Auger electrons can become a major contributor to the therapeutic effect of I-125-NM404.

  The iodine-125 isotope is used as a formulation in this application. Because it exhibits favorable characteristics for radiotherapy of cancer, its properties are listed below:

(1) Half-life of radioisotope: 59.43 days (2) Energy of gamma ray: 35.5 keV
(3) X-ray energy: 27.5-31.7 keV
(4) Radiation half distance: 0.02 mm lead; 2 cm in tissue

Auger electron (1) Radiation energy: 1 keV
(2) Number of Auger electrons: 21 or less with respect to attenuation of gamma rays (3) Half distance of radiation: 1 to 10 nm

  Iodine-125 is very useful for in vivo biodistribution studies and dosimetric extrapolation, for diagnostic imaging, but for the tissue concentration of NM404 by whole body plane imaging or in vivo scintigraphy. It should be noted that it is not well suited for any of the quantifications. However, iodine-124 provides excellent properties for quantitative determination of tissue concentration in vivo. Thus, 124-I-NM404 will be found to be useful for in vivo pharmacokinetic and biodistribution studies. However, 124-I-NM404 has no radiotherapeutic effect and iodine-131 emits both beta and gamma rays that produce a therapeutic effect. Although I-131-NM404 can potentially be used for radiation therapy, we believe that I-125 is the more optimal isotope. This is because it is assumed that the low energy radiation has a shorter radiation half distance than I-131 and therefore will be less damaging to healthy tissue. Therefore, I-125-NM404 was used in all clinical radiotherapy studies conducted to reduce the possibility of causing collateral damage to healthy tissue.

  Because it has a physical half-life of 60 days and emits low energy 35 keV photons, iodine-125 is suitable for imaging experiments in mice and rats. Iodine-125 also provides a therapeutic effect when used on permanent prostate brachytherapy implants (“brace brachytherapy seeds”). The main advantage of 125I is that all photons are low energy, ensuring that the normal tissue surrounding the tumor is exposed to a very limited extent. The main difference between brachytherapy seeds containing iodine-125 and I-125-NM404 is the effect of Auger electrons. Since brachytherapy seeds have a metal capsule around iodine-125, only low energy gamma rays and X-rays have therapeutic value, and Auger electrons are excluded by the metal capsule. In contrast, I-125-NM404 is incorporated into the cancer cell membrane (including its nuclear membrane), and Auger electrons can mainly contribute to the therapeutic effect.

  Although iodine-131 is used with an excellent effect in the treatment of thyroid cancer, a significant difficulty with iodine-131 is the release of high energy gamma rays. That release can actually expose nearby surrounding tissue to a greater amount of radiation than occurs with iodine-125.

Dosimetry

  Dosimetric evaluation of I-125-NM404 in adult female patients was calculated based on SPECT imaging data using female tumor-free rats after administration of I-131-NM404. The results are listed below:

MIRD extrapolation of 125 I-NM404 rat tissue distribution data initially provided a dose limited to 5 Rad to the 2 mCi adrenal and bladder walls of ¹³¹ I-NM404. Therefore, we conducted a preliminary pharmacokinetic study at 1 mCi in human lung cancer patients. Similar extrapolation calculations for ¹²⁴ I-NM404 dosimetry again provided similar 2 mCi dose levels based on the dosimetry predicted for the adrenal and bladder walls. Preliminary human data (5 patients), however, is very low in any abdominal organ (including bladder and adrenal glands) except for the liver which returned to background levels within 11 days Agent uptake and retention are shown. Although these results are not yet complete, the ¹²⁴ I-NM404 dose that can be tolerated in practice is likely to exceed 4 mCi.

  As a result of these dosimetric evaluations, the following points should be considered:

  These results were based on the worst case scenario of no NM404 excretion from the body. Dosimetry data was calculated with SPECT imaging in rats using I-131-NM404 and then converted to I-125-NM404. The adrenal glands are considered to be dose limiting or critical organs for radiation exposure. In the SPECT imaging experiment, the rat thyroid was not blocked. Therefore, thyroid calculations should be evaluated with caution.

Pharmacology overview

  A comprehensive overview of the synthesis and biological properties of NM404 can be found in US Patent Application No. 60 / 593,190 (filed December 20, 2004), US Application No. 10 / 906,687 (March 2, 2005). And US Provisional Application No. 60 / 521,166 (filed Mar. 2, 2004), all of which are incorporated herein by reference for all purposes.

  Our approach is to design a small molecule carrier molecule, which has a unique biochemical or pharmacological property that exhibits a high degree of tissue or tumor selectivity. Utilizing, diagnostic or therapeutic probes can be selectively delivered to the desired target tissue.

  A variety of animal and human tumors were first observed to contain naturally occurring ether lipids in the cell membrane at much higher concentrations than normal tissues. It was hypothesized that phospholipid ether analogs could accumulate in tumor cells due to their low ability to metabolize these lipids. The inventors sought radioiodinated phospholipid ether (PLE) analogs as potential tumor selective contrast agents. Some of the PLE analogs showed a remarkable universal ability to localize selectively in a wide variety of transplanted rat tumor models, transplanted mouse tumor models, and transplanted human tumor models. There was also a thing.

  A promising hypothesis is that phospholipid ethers are trapped at the tumor membrane because phospholipid ethers cannot be metabolized and removed. In fact, analysis of tumors after administration of radioiodinated phospholipid ether showed only the presence of intact drugs, while analysis of normal tissues (liver and muscle), urine and stool showed only metabolites. It was revealed. Thus, the basis for this targeting concept is the difference in the different clearance rates of phospholipid ether analogs from normal cells versus tumor cells.

All clinical studies of the first produced PLE analogs:

  Phospholipid ethers can be easily labeled with radioiodinated iodides using radiolabeling methods developed in the laboratory. The iodophenyl phospholipid ether analogs are specifically designed so that the radioactive iodine attached to each molecule is stable against easy deiodination in vivo. It has been found that whatever the chemical modification of the phosphocholine moiety and whether the iodophenylalkyl moiety chain length is less than 8 methylene, tumor uptake is reduced or eliminated. We are currently synthesizing 20 radiolabeled PLE compounds and testing them in vitro and in vivo. Two of these, NM294 and NM324 [12- (3-iodophenyl) -dodecyl-phosphocholine], first showed the greatest promise in animal tumor localization studies. These prototype compounds were labeled with iodine-125 and selectively localized in the tumor over time in the animal tumor model described below; 1) Walker 256 carcinosarcoma-bearing Sprague-Dawley rats; 2) breast tumors Carrying Lewis rats; 3) Dunning R3327 prostate tumor-bearing Copenhagen rats; 4) Vx2 tumor-bearing rabbits; and 5) Human breast cancer (HT39), small cell lung tumors (NCI-69), colorectal tumors (LSI 74T), ovarian tumors (HTB77IP3), and athymic mice carrying melanoma tumors. Optimal tumor localization of these agents takes from 1 to several days due to more rapid clearance of radioactivity from normal tissue compared to tumors.

  The specific PLE compounds mentioned in the following paragraphs are shown in FIG.

  Clinical evaluation of NM324: The first produced compounds, NM324 and NM294, showed similar animal tumor localization properties, but NM324 is easily synthesized chemically and is therefore a major compound in the first clinical study Selected as. Although tumors were detected in images obtained in several human lung cancer patients, the images were complicated by the high radioactivity of the liver (FIG. 3).

Second production of PLE analogs: To reduce liver uptake and increase plasma phase, we tested nine structural analogs of NM324 to determine the ratio of tumor to background tissue. Identified drugs that reduced liver uptake. A new PLE analog was synthesized for initial contrast analysis in Dunning R3327 prostate tumor-bearing Copenhagen rats and radiolabeled with ¹²⁵ I. Based on this initial screen, NM404 not only exhibits much lower liver activity than the precursor NM324, but also maintains an increased residence in the tumor (FIG. 4. NM404 is therefore Selected for further imaging and biodistribution analysis in various animal tumor models.

  Therefore, NM404 is currently being evaluated in over 20 xenografts and spontaneous animal tumor models, as shown in Table 2. In all tumor models, this drug showed significant uptake and retention in the tumor regardless of location. Although tumor retention can be explained by the lack of metabolic phospholipase enzymes in tumor cell membranes, the exact mechanism of uptake in tumor cells is unknown. In addition, we know that this drug is not localized to benign intestinal adenomas (polyps), where we further evaluate the propensity of agents to be localized in the middle stage of tumor formation, the hyperplastic stage. It is desirable. We are currently evaluating the selectivity of NM404 in a unique mouse tumor model developed with Wisconsin, where both pre-neoplastic hyperplasia and malignant adenocarcinoma are naturally occurring in the mammary gland. It is formed. Preliminary results have resulted in this model showing that this drug is not taken up and retained in pre-neoplastic lesions. Therefore, this drug is thought to be retained only by malignant tumor cells. If this initial observation is validated, through genomic and proteomic analysis, we can identify major genetic differences between malignant tumors and non-malignant progenitor cells. This identifies completely novel molecular therapeutic targets that can be universal for all types of cancer.

  The table below summarizes the various cancer types and tumor lines evaluated for accumulation by NM404.

  Tissue distribution and dynamics

  Consistently, NM404 was found to be retained in tumor tissue for a long and extended period. The concentration in the tumor is almost stable for weeks after NM404 administration, indicating that it is slowly cleared from the cancer tissue over time. In contrast, NM404 is excreted from normal tissue within a few days and reaches very low levels.

  Furthermore, NM404 was designed to have a long half-life in blood. This ensures up to 10% to 25% uptake of the increased exposure of NM404 to the tumor tissue and the injected dose into the tumor tissue. We believe that it is very important for radiotherapy compounds that the majority of the injected dose accumulates in the tissue of interest. As a result of the long half-life in blood, NM404 accumulates continuously in tumor tissue over time. An example of this pattern is provided in FIG. This can lead to a delay in the onset of therapeutic effect by the accumulation of NM404 in the tumor tissue lasting days or weeks.

  Blood plasma dynamics

  The first generation prototype compound, NM324, was found to have a plasma elimination half-life of 2.43 hours in rats. In contrast, the major compound NM404 has an elimination half-life of approximately 209 hours in rats (the half-life of the distribution phase is 4.86 hours).

Radiation therapy study of I-125-NM404

^During tumor uptake and retention studies in mice using “contrast” doses of ¹²⁵ I-labeled NM404 (15-20 μCi / 20 g mice), some clear therapeutic response was observed (results not published) ). In the Apc ^Min l + mouse breast cancer model, tumor growth remains stopped after a single intravenous injection of NM404. Some of these animals lost all of their hairs larger than breast cancer about 8 days after injection. In addition, these mice also become intestinal tumors, usually suffering from intestinal bleeding that results in severe anemia, which leads to whitening of the mouse's feet. Dr. Moser noted that the paw of these mice returned to pink about 5 days after a single injection of NM404. The final dissection of these animals noted that only about 20 expected intestinal tumors remained at this age, usually very little, if any, at all. The phenomenon of “white to pink paw” was also observed in the isolated but more malignant mouse intestinal adenocarcinoma model. Here, the dissection was 12 days after administration of NM404, again revealing that most if not all of the expected bowel tumors were gone. After 21 days in both intestinal models, animals that received NM404 lived longer easily than untreated littermates. Another compelling example of tumor regression is illustrated in FIG. Two littermates were each inoculated with SCC1 and SCC6 xenografts on the left and right flank, respectively. One mouse received a single injection of ¹²⁵ I-NM404 (20 μCi). Mice that did not receive NM404 died 21 days later, but the tumors in the treated mice regressed significantly and the animals were very healthy even 80 days after injection. These matched findings were reconfirmed in two separate age-matched groups, each using more than 6 mice. ^Although these observations with ¹²⁵ I-NM404 are uncertain in this respect, these observations strongly suggest potential for radiotherapy applications, particularly when labeled with iodine-131. it is conceivable that. A continuous quantitative tumor uptake and retention study in several animal tumor models is needed to begin a comprehensive dosimetric analysis of this agent to assess its true radiotherapy potential. Provide sufficient data.

With a physical half-life of 60 days and low energy 35 KeV photon emission, iodine-125 is suitable for imaging experiments in mice and rats. Iodine-125 also has therapeutic properties. In one imaging experiment (FIG. 6), two nude mice were each inoculated with SCC1 and SCC6 tumor cell grafts of the subcutaneous squamous cell line on the opposite flank, respectively. SCC1 and SCC6 cells were used because one was radiosensitive to the other. Fourteen days after the average tumor size reached 0.5 cm in diameter, one mouse received 20 μCi of I-125-NM404 and the other mouse received an equal dose of unlabeled NM404. Mice that received unlabeled cold compound were euthanized 20 days after injection because both tumors reached the limit of termination size as defined in the animal use protocol. Over a period of several weeks after a single injection of a contrasted dose of NM404, both tumors in ¹²⁵ I-NM404 mice regressed dramatically and unexpectedly (FIG. 6). The mice never reached terminal tumor size, and in fact the mice were euthanized after 90 days to collect histological sections.

Preliminary report of ongoing tumor treatment research

  Preclinical studies-preliminary results:

Effect of a single injection of I-125 labeled SF404A in the A549 SCID mouse model

Objectives and rationale : To determine the effective dose for radiotherapy using a single injection of I-125 labeled SF404A in a xenograft tumor model

Tumor model:

A549 (from human non-small cell lung cancer, NSCLC, ATCC) was maintained in Ham's F-12K medium supplemented with 10% fetal bovine serum. Tumor cell suspension (1 × 10 ⁶ cells in phosphate buffered saline) is injected subcutaneously into the right flank of female SCID mice (6-8 weeks, CB-17 / lcrHsd-Prkcd ^scid , Harlan). The animals had free access to food and water and were monitored for tumor growth and animal weight. When reaching a diameter of 4-5 mm, the mice are divided into 6 groups for therapeutic studies.

Dosage :

  One injection of SF404A on day 0

Group :
(1) 5 study groups: 4 treatment groups + 1 control group (2) N = 6 per group
(3) Four exact dose levels: 50 μCi, 150 μCi, 250 μCi and 500 μCi for each mouse
(4) Control group administered with an equal dose of NM404:
(5) Control: each mouse 0 μCi (“cold” NM404)

Effectiveness assessment :
(1) Tumor size (caliper) measured once a week
(2) Survival rate (3) Overall status (activity, alertness, mobility)
(4) Up to 10 weeks after injection or the animals in the control group will not survive at all, whichever comes first (5) Assessment of the pathological tissue of the tumor or residual tumor site

  Tumor photographs at day 0, day 30 and day 60 for each animal.

  The purpose of this study was to determine the effective dose for radiotherapy using a single injection of I-125-NM404 (formulation SF404A) in a xenograft tumor model.

The tumor model used in this study was A549, a human non-small cell lung cancer (NSCLC) obtained from ATCC (Manassas, VA). Tumor cells are maintained in HamF12K medium supplemented with 10% fetal bovine serum. Tumor cell suspension (1 × 10 ⁶ cells in phosphate buffered saline) is injected subcutaneously into the right flank of female SCID mice (6-8 weeks, CB-17 / lcrHsd-Prkcd ^scid , Harlan). Animals have free access to food and water. When the tumor diameter reaches 5-10 mm, the mice are listed in the study directory.

  A single intravenous injection of SF404A was performed on day 0. As a control group, a cold compound (C-NM404) was injected. Treatment groups were injected with I-125-NM404.

  The study included 5 groups; 4 treatment groups (n = 6 / group) and 1 control group (n = 9). The iodine dose in the treatment group was 50 μCi, 150 μCi, 250 μCi and 500 μCi per mouse. An equal dose of C-NM404 (0 μCi) was administered to the control group.

Assessment of treatment effect and subsequent assessment:
(1) Tumor size (caliper) measured once a week
(2) Survival rate (3) Overall appearance (activity, concentration, mobility)
(4) Digital photograph of tumor-bearing mice.

Assessments were made either after 10 weeks of injection or until either of the control group animals no longer survived. Histopathological assessment of the tumor or residual tumor site was performed at the end of the study.
(1) 50 μCi group: 6 animals were registered;
(2) 150 μCi group: 5 animals were registered;
(3) 250 μCi group: 6 animals were registered;
(4) 500 μCi group: 6 animals were registered.

As part of this preliminary report, we add the following animals in the analysis.
(1) Control: n = 7
(2) 50 μCi group: n = 4
(3) 150 μCi group: n = 5
(4) 250 μCi group: n = 6
(5) 500 μCi group: n = 5

The baseline tumor volume (mm ³ ) for each group is:

Met.

  Since the tumor volume in the control group and 150 μCi group was substantially larger than the other groups and larger than previously planned, re-registration of both groups with smaller tumor sizes was begun. As far as the tumor volume of the 500 μCi group is concerned, we believe that the study may be biased against this group. Due to necrosis and inefficient blood supply, larger tumors cannot respond well to I-125-NM404 treatment.

  The average tumor volume for each of the following groups was recorded over a 10 week assessment period:

  This is also shown in FIG.

  In short, control animals show rapid tumor growth over a 10 week assessment period. This confirms that the compound itself, C-NM404, has no substantial effect on tumor growth. The 50 μCi dose group does not show any difference from the control animals and therefore this is not considered an effective dose in this animal model. The 150 μCi dose group similarly showed no therapeutic effect, but this dose group started with an abnormally sized tumor on day 0 (as pointed out earlier). However, this dose group may show different results from this 150 μCi dose group with smaller tumors.

  Preliminary data indicate that both 250 and 500 μCi show a substantial and increased therapeutic effect. The tumor volume is stable and the same tumor is considered “collapsed” (the tumor surface has collapsed, shown in FIG. 7). Furthermore, the hair on the tumors fell off, indicating that there was substantial accumulation of radioactivity in these tumors.

Other ongoing preclinical tumor treatment studies

Effect of split dose vs. single injection of I-125 labeled SF404A in the A549 SCID mouse model

  This preclinical study assesses splitting a 150 μCi dose by injecting a 50 μCi dose for 3 weeks instead of the single 150 μCi dose given in the previous study.

Objectives and rationale : To determine the radiotherapeutic effect of a single injection of an equal total dose to a divided dose of I-125 labeled SF404A in a xenograft tumor model. This study is in addition to CTLR-Pre-05-001, which includes a single dose of 150 μCi used for comparison.

Tumor model

A549 (from human non-small cell lung cancer, NSCLC, ATCC) is maintained in Ham F12K medium supplemented with 10% fetal bovine serum. Tumor cell suspension (1 × 10 ⁶ cells in phosphate buffered saline) is injected subcutaneously into the right flank of female SCID mice (6-8 weeks, CB-17 / lcrHsd-Prkcd ^scid , Harlan). To do. Animals have free access to food and water, and tumor growth and animal weight are monitored. When the diameter reaches 4-5 mm, the mice are divided into 6 groups for treatment studies.

Dosage :

  On day 0, a single injection of I-125-SF404A vs. an injection of divided doses of I-125-S404A

Group :

2 study groups: 1 divided dose, 1 single injection (1) N = 6 per group
(2) 150 μCi per mouse (3 × 50 μCi) by split injection, 1 week interval from day 0 (3) 150 μCi per mouse by single injection

Assessment of effectiveness :
(1) Tumor size (caliper) measured once a week
(2) Survival rate: 10th week after injection or until the animal is no longer alive in any group, whichever comes first (3) Overall appearance (activity, concentration, exercise sex)
(4) Histopathological assessment of tumor or residual tumor site (5) Photographs of tumors on day 0, day 30 and day 60 for each animal.

Effect of a single injection of I-125 labeled SF404A in a PC-3 (prostate cancer) SCID mouse model

Objectives and rationale : To determine the effective dose for radiotherapy using a single injection of I-125 labeled SF404A in a prostate cancer xenograft variable tumor model.

Tumor model :

PC-3 (from human prostate cancer, ATCC) is maintained in Ham F12K medium supplemented with 10% fetal bovine serum. Tumor cell suspension (1 × 10 ⁶ cells in phosphate buffered saline) was injected subcutaneously into the right flank of male SCID mice (6-8 weeks, CB-17 / lcrHsd-Prkcd ^scid , Harlan). To do. Animals have free access to food and water and monitor tumor growth and animal weight. When the diameter reaches 4-5 mm, the mice are divided into 6 groups for therapeutic studies.

Dosage :

  One injection of SF404A on day 0

Group :
(1) 5 study groups: 4 treatment groups + 1 control group (2) N = 6 per group
(3) Four acute dose levels: 50 μCi, 150 μCi, 250 μCi and 500 μCi per mouse
(4) Control group dosed with equal amount of NM404; 0 μCi per mouse (“cold” NM404)

Effectiveness assessment :

  Tumor size (caliper) measured once a week

Survival rate : 10 weeks after injection or until no animal in any group survives (1) Overall appearance (activity, concentration, motility)
(2) Histopathological assessment of tumor or residual tumor site (3) Tumor photographs on day 0, 30 and 60 for each animal.

  This preclinical study repeats the previous study, which is a single injection study, but uses a different tumor model.

Clinical evaluation for prostate and NSCLC imaging using NM404, a phospholipid ether analog

A safety assessment is conducted in normal male subjects and healthy normal female subjects, and a dose level of 3 μg / kg of C-NM-404 may be safe to administer to male subjects. It was found. This dose is 10 times the 0.3 μg / kg dose of ¹³¹ I-NM404 expected to be used for imaging in patients with metastatic prostate cancer. The 3 μg / kg dose level of C-NM404 is safe to administer to female subjects. This dose is 10 times the 0.3 μg / kg dose of ¹³¹ I-NM404 expected to be used for imaging in patients with metastatic lung cancer.

NM404 contrast characteristics in patients with NSCLC

  Metastatic non-small cell lung cancer subjects:

Written informed consent is obtained. A 0.3 μg / kg dose of ¹³¹ I-NM404 is infused over 10 minutes. An aqueous solution containing ¹³¹ I-NM404 is prepared using sterilization techniques in the laboratory of Dr. Jamesy Weichert, University of Wisconsin. Preparations formulated by Nuclear Pharmacy are proven to be sterile and pyrogen-free. Subjects are monitored for side effect reactions during and after infusion. Vital signs are checked at 60 minute intervals immediately after infusion and until 4 hours after injection. Throughout this period, vital signs are observed with respect to adverse events. Subjects return 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours and 30 days after infusion. NM404 PET scans are performed at 4 hours, 8 hours, 24 hours, 48 hours and 96 hours after injection. At these time points, vital signs are monitored. Record any side effects that occur in the subject. To reduce thyroid exposure to free radioactive iodine, all subjects will receive SSKI oral solution starting 7 days prior to ¹³¹ I-NM404 infusion and continuing for 7 days. Serum pharmacokinetic results were shown before injection, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 240 minutes, 360 minutes after injection, and 24 hours, 48 hours after injection. 72 hours and 96 hours later. Sequential 24 hour (0 to 24, 24 to 48, 48 to 72, and 72 to 96 hours after injection) urine collections are obtained.

  Plasma pharmacokinetics supported an elimination half-life of an average of 113.1 hours (SEM = 7.9 hours) for NM404.

  An average of 3.4% of the injected dose (range 0.9% to 9.8%) was excreted via the kidney 96 hours after intravenous administration of NM404. A dose level of 0.3 μg / kg of 131I-NM404 was safe to administer to subjects with advanced non-small cell lung cancer. The excretion plasma half-life of NM404 was found to be 113.1 hours, and excretion into the urine was found to be approximately 3.4% within 96 hours after injection.

Prostate Cancer The present invention provides preliminary data regarding the use of NM404, a second generation PLE analog, in imaging patients with prostate cancer. This drug is currently being investigated at the University of Wisconsin, is selectively retained in tumors at high levels, and has high sensitivity and specificity in preclinical models. The drug passed acute poisoning studies in both rats and rabbits even at doses exceeding 1000 times the expected human contrast dose. And to demonstrate safety, 10 normal volunteers were administered unlabeled drug at 10 times the expected contrast dose at the University of Michigan and the University of Wisconsin. The inventors finally demonstrated that contrast using NM404 is as sensitive as contrast using FDG, and contrast using NM404 is also more specific and can be used for therapy. And because of the relatively long half-life, it can be used in virtually all PET facilities regardless of location.

^The biodistribution, kinetics, optimal imaging time and dosimetry of ¹³¹ I-NM404 are currently being evaluated in pilot studies in patients with lung cancer (NSCLC) at UWCCC. The drug was also evaluated preclinically in a metastatic prostate tumor model, in which lymph node metastases were clearly delineated by scintigraphy after intravenous administration of NM404, but tracers were not It did not stay properly. The selectivity of NM404 for malignant cells is particularly relevant for 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. Moreover, selective uptake and increased retention by tumor cells support the role of NM404 as a tumor selective diagnostic and therapeutic agent.

Preliminary results provide preliminary data from continuous studies designed to more accurately assess the predictive power of NM404 for staging and / or response to treatment in prostate cancer. Furthermore, because NM404 has high uptake in tumors, this drug also has the potential to be developed as a therapeutic agent when combined with high doses of ¹³¹ I, ¹²⁵ I, another halogen astatine. Iodine-125 is desirable, particularly in prostate cancer patients, due to the short Auger electron orbital length in the tissue and theoretically minimizes radiation effects in nearby normal tissues such as the rectum. In the therapeutic sense, the 60-day half-life of iodine-125 is in excellent agreement with the increased tumor retention properties of NM404.

  Targeting tumor cells

  One approach to the development of sensitive and more useful imaging tests is to design carrier molecules that have the ability to selectively deliver radiopharmaceutical probes to the desired target tissue. The approach takes advantage of the inherent biochemical and pharmacological properties of phospholipid ether analogs (eg, NM404) that exhibit a high degree of tumor selectivity. Phospholipids are an essential component of cell membranes, where phospholipids provide structural integrity and are highly associated with many cell signaling processes. Phosphatidylcholine is well known as lecithin and is one such example. Phospholipid ethers, on the other hand, represent a minor subclass of phospholipids that are also present in the membrane. As the name suggests, these lipids contain an ether linkage rather than an ester linkage at the C-1 position. Platelet activating factor (PAF) represents one of the well-known phospholipid ethers. As mentioned above, the retention of PLE in tumors is due to different PLE clearance rates in normal cells versus tumors.

  An ongoing experiment was undertaken to identify PLE compounds with excellent tumor localization and background clearance properties while initiating human pharmacokinetic studies with the prototype agent NM324. Based on this study, NM404 [12- (4-iodophenyl) -octadecylphosphocholine] is used for enhanced ability to localize in tumors, increased metabolic clearance from the liver, and long plasma half-life. Selected. In a major experiment demonstrating NM404's ability to localize to metastases, lymph node metastases were clearly delineated by scintigraphy in a metastatic prostate tumor model after intravenous administration of NM404, but tracers It did not stay in the knot.

According to the comparative scintigraphic contrast results in SCID mice bearing PC-3 prostate tumor (for NM324 and NM404), the ratio of tumor to normal tissue was large, and NM404 in the background abdomen and liver It became clear that the radiation of (1) decreased significantly (FIG. 19). This drug is currently being evaluated in 27 animal tumor models and it is clear that once the drug enters the tumor cells, it reaches the end of metabolism and is trapped. Increased tumor retention of this drug has been demonstrated in human adrenal tumor xenografts implanted in SCID mice (FIG. 20). ^{Using 125} I-labeled NM404, we were able to image breast and prostate tumors in mice for more than 60 days. Increased tumor retention properties significantly enhance the radiotherapeutic effect of the drug.

  Recent imaging and biodistribution studies conducted in rodent models aim to determine uptake characteristics in a variety of xenografts and spontaneous (endogenous or transgenic) tumor types, It is summarized. These agents showed selective localization and increased retention in all malignant tumors, regardless of the anatomical locations studied to date (including lymph nodes).

Preliminary scintigraphic imaging results with NM404 in prostate tumor-bearing mice: In a preliminary study showing that NM404 is localized in the prostate tumor of mice, TRAMP mice were injected with tail vein injection of ¹²⁵ I-NM404 (15 μCi). From day 1 to day 8 after, scanning with a bioscan AR-2000 radiation TLC scanner (corrected in the laboratory for mouse imaging). To avoid tissue weakness associated with low energy iodine-125, prostate tumors were immediately removed after in vivo imaging of anesthetized mice and the same scanner (high resolution 1 mm collimator and 2-D acquisition) And provided with analysis software) ex vivo imaging (FIG. 21). Although the number of prostate tumor-bearing animals was small (n = 4), preliminary imaging and tumor-to-background data show NM404 uptake in prostate tumors, not the benign hyperplasia seen in this model. It was. In an attempt to simulate bone metastasis, another experiment was performed. In this experiment, human PC-3 tumor cells were implanted in the tibia of immunodeficient nude mice (n = 6) according to a recent report. Following tumor cell inoculation, mice were continuously scanned with high-resolution micro-CT to monitor bone tumor progression (Figure 22). Once the bone degradation was detected, iodine-125 labeled NM404 was administered intravenously via the tail vein. Mice were scanned for radioactivity by micro-CT 4 days after NM404 administration. The radioactivity scan was fused with a micro CT scan (FIG. 23) to confirm the radioactivity of NM404 and the location of the tumor.

Biodistribution data Extended biodistribution data for the prototype agent ¹²⁵ I-NM404 has been previously reported in several tumor models. Tumor to blood ratios over 8: 1 were seen with lag time after injection. For example, in a rat breast cancer model, the tumor to normal tissue ratio reaches a maximum at 96 hours, when the tumor to blood ratio is 8.6 and the tumor to muscle ratio is 20: 1. there were. Furthermore, the biodistribution of PLE-related radioactivity is heterogeneous in tumors, as demonstrated by microautoradiogram studies, indicating that the PLE radioactivity is in the outer region rather than the central necrotic region. It is present exclusively in living tumor cells located towards the. Comparative biodistribution data for NM324 and NM404 in SCID mice was obtained in prostate and A549 lung cancer tumor models. These studies reveal that for NM404, the ratio of tumor to normal tissue is large and that the tumor uptake exceeds 25% of the injected dose, thus this study shows the biodistribution of PLE analogs in humans. Support the desire to test.

Relatively short-lived nuclides (eg, ¹⁸ F or ^99m Tc) are now practical for labeling NM404 because isotope-selective PLE analog tumor targeting strategies are believed to involve selective tumor retention over time Not right. It can also prove that iodine-123 is suitable for this drug when scanning in 3D SPECT mode, as it has a half-life of 13 hours and optimal imaging properties. Imaging with iodine-123 requires further investigation. Tumor localization of PLE analogs appears to occur within hours of injection (NM324 images were obtained 6 hours after injection in lung cancer patients that showed intense uptake in lung tumors in previous studies) . On the other hand, planar two-dimensional imaging, as done recently in humans with iodine-131, has a lag time to allow background activity to be removed from nearby normal tissue and blood. is necessary. It seems that earlier imaging may be possible when scanning with PET and 3D SPECT. In that case, nearby radioactivity is less subject to interference due to the three-dimensional nature of these therapeutic application modes. In organs that remain essentially low in background radioactivity (eg, the brain), gamma-radiation isotopes such as iodine-123 (this provides the same image after injection, in addition to providing a more beautiful image) Image acquisition is possible). Although the use of other isotopes may eventually prove to be suitable for use with NM404, the current focus is on this drug based on the current success of tumor imaging using a hybrid PET-CT scanner Will develop the PET imaging ability of The superior diagnostic accuracy made by biochemical or functional tumor imaging PET agents combined with the precise anatomical accuracy provided by CT is currently the gold standard for tumor imaging. However, it is very convenient to label the PLE analog with iodine-124, a relatively new PET isotope, and its physical half-life (4 days) is good for PLE tumor uptake and retention kinetics. Match. Labeling NM404 with iodine-124 represents a natural extension of previous work with nuclides that emit gamma rays. It has been shown that PET imaging with ¹²⁴ I exceeded 40 times the sensitivity of planar ¹³¹ I-gamma scintigraphy. PET, unlike traditional gamma camera imaging, also provides significant resolution enhancement, contrast quantification and three-dimensional capabilities. To overcome the limitations inherent in planar scintigraphy in current lung cancer imaging studies, labeling NM404 with iodine-124 based on ¹³¹ I-NM404's preliminary success, and tumor detection with PET It is now essential to evaluate the effect.

The use of tumor tracers (eg, ⁶⁷ Ga-citrate and ¹⁸ F-FDG) is limited due to the lack of specificity that distinguishes neoplasia from inflammation. This lack of specificity is an important clinical problem in patients with cancer. However, preliminary studies of PLE analogs provide a promise to overcome this limitation. Previous experiments conducted in rats revealed no uptake and retention of NM324 in granulomas induced by carrageenan. Gallium citrate, however, was used as a control in this study, but it did not actually accumulate significantly in granulomatous lesions. Thus, this preliminary finding that PLE analogs are not clearly localized in inflammatory lesions further justifies the need for evaluation of this drug in human cancer patients. Although FDG-PET has paved the way for hybrid imaging methods, the lack of tumor cell specificity always limits the diagnostic effectiveness. Novel molecular targeting agents, such as NM404, can be used in universal tumors regardless of the location and selectivity of malignant tumor cells (but not in terms of selectivity to non-inflammatory or proliferative lesions). Shows uptake and selective retention, showing significant improvement in cancer detection and characterization.

Whereas planar nuclear medicine imaging techniques have historically made acceptable 2D images, this mode of treatment application lacks tomographic capabilities and poor image quantification. ¹²⁵ I-NM404 was suitable for preliminary test scintigraphic imaging and tissue distribution studies in rodent tumor models, and ¹³¹ I was suitable for phase 1 safety and pharmacokinetic assessment in human lung cancer patients . However, both are not optimal for quantification with in vivo human imaging. PET imaging using iodine-124 is relatively new and commercially available positron isotopes with a 4-day half-life alleviate many of the problems associated with planar imaging. Transferring NM404's unique tumor imaging capabilities to PET scanning is now the main objective of our laboratory. This is because iodine-124 came on the market in the second half of last year. When the tumor specificity of NM404 against malignant tumors found in mouse models has been confirmed in humans, we have found agents that have tumor specificity over FDG despite lacking inflammatory site localization properties. Have. In addition, the drug can be manufactured in one facility and can be transported to virtually anywhere in the world because it has a half-life of 4 days.

The inventors have recently successfully radiolabeled NM404 twice with iodine-124 provided by a private manufacturer (Eastern Isotopes). Radiochemical yields (> 60% average separation yield,> 99% purity) are much higher than those normally obtained with commercial commercial sources of iodine-125 or 131 sodium iodide. It was very similar. The PET imaging properties of ¹²⁴ I-NM404 in a rat CNS-1 brain tumor model were tested (FIG. 24). The imaging time in this preliminary study was limited 24 hours and 4 days after injection due to the availability of the microPET scanner. MicroPET images obtained 24 hours after intravenous injection of ¹²⁴ I-NM404 supported MRI images with enhanced contrast and showed tracer intensity uptake in brain tumors. The tracer is hardly or not taken up by the surrounding native brain tissue. This study represents the first PET imaging obtained with NM404 and demonstrates the ability to efficiently radiolabel, purify, and formulate NM404 for PET imaging. These compounds can then be used for extended use of NM404PET in human cancer patients.

By PET-CT in patients with metastatic prostate cancer proven by radiography ¹²⁴ Determine tumor uptake and retention characteristics of INM404

  Purpose and rationale

  The assessment of whether radioiodinated NM404 is capable of selective detection of known metastatic foci and whether it can be compared to conventional radiotherapeutic therapeutic applications is discussed in a later paragraph. The inclusion diagnostic criteria in this study consisted of 15 patients with metastatic prostate cancer, at least 5 patients have soft tissue prostate cancer metastasis and at least 5 patients have bone metastasis. These metastases can be identified by conventional radiological research, including CT scans and bone scans. Following patient enrollment, uptake of radiolabeled NM404 is measured by I-124 isotope PET-CT scanning. This correlates with the lesions revealed by radiographs detected by other conventional staging studies of the patient.

  Method

Synthesis, Radiolabeling and Formulation: Radioiodination of stable NM404 with ¹²⁴ I-sodium iodide is routinely achieved by modification of the ammonium sulfate-mediated isotope conversion reaction reported by Mangner, Recently optimized for NM404. The conversion reaction methodology has been used effectively in the first human trial with NM324, a precursor of NM404, and is currently used in preclinical studies and human lung cancer trials. Temporarily, a 2 ml glass vial is filled with 10 mg ammonium sulfate dissolved in 50 μl deionized water. Fourteen 2 mm chemically resistant glass beads are added, a Teflon lined diaphragm and screw cap are attached, and the vial is gently swirled. NM404 stock solution (20 μg) (in 20 μl ethanol) is added, followed by sodium iodide aqueous solution (124, 1-5 mCi) in less than 30 μl 0.01 N sodium hydroxide aqueous solution. The isotope syringe is washed 3 times with 20 μl of ethanol once. Gently swirl the reaction vial. A 5 ml disposable syringe containing glass wool is attached at the outlet of the needle in series with another 5 ml charcoal-filled syringe. The glass wool syringe serves as a concentration chamber for collecting evaporated solvent and the charcoal syringe traps free iodide / iodine. The reaction vessel is heated in a heat block apparatus at 150 ° C. for 45 minutes. A volume of 20 ml of air (four) can be injected into the reaction vial with a 25 ml disposable syringe and released through a double trap accessory. The temperature is raised to 160 ° C. and the reaction vial is heated for an additional 30 minutes. After cooling to room temperature, ethanol (200 μl) is added and the vial is swirled. To remove unreacted iodide, the ethanol solution is passed through a pre-equilibrated Amberlite IRA 400-OH resin column. The volume of the eluent was reduced to 50 μl via a nitrogen stream (using a charcoal syringe trap) and the remaining volume was purified on a silica gel column (Perkin Elmer, hexane / isopropanol / water (52:40) for purification. : 8) and 3 μm × 3 cm disposable cartridge column eluted at 1 ml / min). Final purity is determined by TLC (plastic backed silica gel-60 eluted with chloroform-methanol-water (65: 35: 4, Rf = 0.1)). The HPLC solvent is removed by rotary evaporation and the resulting radioiodinated NM404 is solubilized with a 2% pharmaceutical grade polysorbate-20 aqueous solution (compound 0.1 μl / mg). The ethanol is removed under reduced pressure and the remainder is dissolved in sterile water to obtain a final solution containing 2-3% or less of polysorbate-20. Sterilization is achieved by filtering through a sterilized 0.2 μm filter unit. Each solution is tested for pyrogens using the Limulus Ambocyte Lysate test kit. This is the same procedure currently used for the preparation, purification and sterile formulation of I-131 labeled NM404 used in lung cancer patient studies. The drug Master Formulation Card and the Product Preparation Checklist are included in the supplemental part of this proposal. We have performed this radioiodination hundreds of times with either iodine-131 or 125, and generally have a radiation in the range of 60-90% of NM404 with a specific activity greater than 130 mCi / μmol. Chemical yield (purified product isolated) was achieved. This method resulted in sufficient, pure and injectable ¹³¹ I-NM404 for ongoing preclinical and human studies. The present inventors have not failed the labeling to date. In addition, with respect to the current proposal, we have recently successfully radiolabeled NM404 with iodine-124 sodium iodide (5 mCi) obtained from Eastern Isotope in> 60% isolated radiochemical yield. did. Acceptable diagnostic criteria require> 95% radiochemical purity, but since all reaction mixtures have been subjected to preparative HPLC purification, results are typically above 99%.

I-124 dose for human administration: ¹³¹ I NM404 toxicity analysis results, biodistribution, kinetics, optimal imaging time and dosimetry are ongoing in patients with lung cancer (NSCLC) at UWCCC It has been recently evaluated in research. The dose of radioiodinated ¹²⁴ I NM404 is based on MIRD extrapolation of the iodine-131 dosimetry data from this ongoing study. The dose of ¹³¹ I-NM404 is recently calculated as follows: Animal biodistribution data is used to determine the percentage of injected dose / organ at various time points. These animal data are extrapolated to humans using the MIRD format method (MIRDOSE PC v3.1). This method uses standard conversion factors for differences in organ mass and anatomy between rats and standard humans and provides expected human organ doses; Based on the predicted dose, the mCi dose allowed to be injected into humans is determined by the RDRC regulations for specific human tissues as specified in the Federal Register (21 CFR Part 361.1). Determined using maximum legally acceptable dose. For example, whole body, blood, hematopoietic tissue, ophthalmic lens, gonadal-3 rem / dose; other tissues-5 rem / dose. This approach was previously used for clinical use of both NM324 and NM404, resulting in a calculated dose of 1 mCi. New animal data obtained with NM404 (5 rad testicle dose = 2.1 mCi and 5 rad adrenal dose = 2.2 mCi), the starting maximum dose of ¹³¹ I-NM404 is around 1.9 mCi Or twice that of NM324.

Study Procedure: Establish an intravenous line on the arm prior to injection. A tracer dose of ¹²⁴ I-NM404 (1 mCi, actual dose is the result of an ongoing study dosimetry) less than 0.3 μg / kg body weight is infused over 5 minutes. The preparation is sterile, pyrogen-free and contains less than 5% free iodine by thin layer chromatography (usual results are less than 1%). Patients are clinically monitored for adverse reactions during the infusion.

Preliminary clinical safety studies with animal toxicity studies (in this study using more than 1000 times the planned maximum amount of ¹²⁴ I-NM404) and unlabelled compounds (more than 10 times the dose) were conducted without side effects. So we do not anticipate toxicity at contrast doses. However, we recognize that there may be inappropriate allergies or other reactions. The patient's vital signs (pulse, BP, body temperature and respiratory rate) are rechecked immediately after infusion and at 30 and 60 minutes after infusion. The vital signs are then checked every 2 hours for 2 hours.

Baseline PET-CT imaging (full body and selected local bond imaging) for the first 5 patients is obtained 48 and 96 hours after injection. PET contrast is obtained using a GE Clinical GE Discovery LS PET-CT scanner with appropriate attenuation correction and optimized for iodine-124. The 48 and 96 hour time points are based on preliminary data on the optimal contrast time for ¹³¹ I-NM404 in lung cancer. However, the optimal tumor target time for ¹²⁴ I-NM404 in human prostate cancer has not yet been determined.

Accumulation and washout of the ¹²⁴ I-NM404 tracer is recorded with a GE Discovery LS PET / CT scanner or GE Advance PET scanner available in our clinic. To correct for patient localization and attenuation, a CT scan is first performed using a Discovery LS PET / CT scanner; a transmission scan is performed with an Advance PET scanner. Each PET scan is optionally corrected for dead time, attenuation and diffusion. The first scan is performed rapidly 48 hours and 96 hours after the ¹²⁴ I-NM404 injection. A full body scan is performed with increasing scan time (up to 30 minutes beyond the tumor site). The image is reconstructed using an OSEM reconstruction method that interacts with attenuation correction and smoothed with a Gaussian filter.

The evaluation of the data is based on a region-of-interest (ROI) analysis recorded with the PET contrast. The CT obtained by each PET / CT scan is recorded so that the correct part of the PET data can be specified. The ROI is sketched to depict various organs (typically heart, lung, muscle, liver, stomach, spleen, intestine, kidney and bladder) from a diagrammatic point of view. At each ROI and each time frame, the average radioactivity concentration is calculated and standardized uptake values (SUVs) are calculated. Detailed pharmacokinetic analysis is not performed in this study. However, it has been completed in an ongoing lung cancer trial, which is not mentioned here. This is because it is performed in the lung cancer test. In the lung cancer test, PET scans are performed immediately after injecting NM404 in two patients, followed by PET scans at 6, 24 and 48 hours after injection. SUVs are plotted as time-behavior curves. The time course of radioactivity in each ROI is fitted using various kinetic models. This model includes 2-rate constants in a two-compartment model, and 4-rate constants and 3-rate constants in a three-compartment model, and those related to non-linear regression fitting. Non-linear, at least square fitting is done using a modified gradient-expansion algorithm. The best fit is determined by minimizing the χ ² function for the variables in the model parameters. To test the assumption of irreversibly trapped phospholipids in the tumor membrane, for the 3k model, if k ₄ is set equal to 0, the data fit. Furthermore, the Patlak plot is used to normalize tissue radioactivity kinetics relative to plasma radioactivity kinetics.

Statistical considerations: This is feasible to determine the uptake and retention characteristics of radioiodinated ¹²⁴ I-NM404 in tumors by PET-CT in patients with clinical evidence of metastatic prostate cancer (canter) Research. Fifteen patients with metastatic prostate cancer will be collected for Part 1 of this study. Contrast scans are recorded using a general 0-3 step standard: 0 = no perceptible uptake, 1 + = slight perception above background, 2 + = uptake above background clearly 3 + = intensity uptake, much more than the surrounding normal structure (2+ and 3+ are considered abnormal or positive for lesion identification; 0 and 1+ are considered normal or negative for lesion identification). Uptake rates classified as positive for identification of at most 50% lesions are considered clinically inappropriate. Therefore, we test the alternative hypothesis that the percentage is greater than 50%, versus the null hypothesis that the percentage of uptake classified as positive for lesion identification is approximately 50%. We expect uptake ratios classified as positive for lesion identification to be at least 80%. Assuming that the total sample size is 15 patients, the one-sample binomial test with a null hypothesis that the uptake rate classified as positive is approximately 50% is 10 It has a power of 85% to detect a percentage of 80% (with a one-sided test) at the% significance level. A ratio of 90% is detected with a power of 95%. Since the sample size is 15 patients, the proportion of tumors classified as positive for lesion identification has a standard error of at most 13% and the 90% confidence interval for this proportion is wider than 39% There is no.

In patients with clinically narrowed prostate cancer who are candidates for thorough prostatectomy with bilateral pelvic lymph node dissection, ¹²⁴ Determination of specific tumor accumulation and metabolic fate of I-NM404

  Purpose and rationale

  As a second preliminary analysis, malignant and benign prostate and nodular tissues are obtained by thorough prostatectomy and bilateral lymph node dissection and evaluated for radioiodinated NM404 accumulation and NM404 metabolites. The data obtained by this tissue analysis is compared with the results of the final pathological analysis. Ten patients with organ-restricted prostate cancer are planned and registered for a thorough prostatectomy with bilateral pelvic lymphadenectomy. All patients will undergo conventional preoperative staging studies, including CT and bone scans. All patients will also have a NM404 PET-CT scan in the week prior to the planned prostatectomy. Final pathological analysis of resected prostate and lymph glands correlates with NM404 accumulation and metabolite tissue assays, and in patients with local-region metastatic disease, relative to tumor volume in the first specimen and lymph nodes Determine the relationship of symptoms. Despite negative preoperative CT and bone scans, nodular metastases detected by NM404 accumulation are of great clinical importance in terms of improving staging of these patients prior to treatment.

Study Procedure: The imaging procedure in this study is that the patient is imaged only once based on the optimal imaging time identified in the initial protocol (possibly between 24 and 48 hours after injection). Except for the first study. Six prostate biopsy cores were obtained from prostate specimens in the OR immediately after surgical removal from the sex distribution (left and right cusps, mid-gland and basal) Region). These six biopsy cores are evaluated by frozen section pathology to ensure representative sampling of both malignant and benign prostate tissue specimens. A second set of 6 biopsy cores, obtained simultaneously from the same corresponding site in the prostate, is analyzed for uptake of ¹²⁴ I-NM404. Therefore, they are photographed, subjected to high resolution scanning with a Bioscan AR2000 radiation scanner (radioscanner), and also weighed and quantified for radioactivity with a well counter. The radioactivity concentration is determined in% dose / g tissue sample based on comparative examples. These results are compared with histological results to ensure localization in the tumor as compared to surrounding tissues not involved. A core biopsy is labeled with the patient's study identification number and the resulting site in the prostate.

The final symptom of any lymph node in which a metastatic prostate tumor is present also correlates with the ¹²⁴ I-NM404 uptake signal detected by pre-operative PET-CT scanning. Vital signs are obtained after infusion of ¹²⁴ I-NM404.

  Statistical considerations: Ten high-risk patients with clinically localized prostate cancer will be collected for Part 2 of this study. Part 2's null hypothesis is that the log ratio of NM404 accumulation in tumor tissue is zero relative to the normal surrounding tissue of clinically positioned prostate patients. The alternative hypothesis is that the log ratio of NM404 accumulation is greater than zero. A tumor to normal tissue (T / N) ratio of at least 5: 1 is considered clinically relevant. Based on past experiments on NM404, the standard deviation of the log proportion of NM404 accumulation is expected to be between 1.5 and 2.0. Since the sample size is 10, a one-sided t-test with a one-sided 10% significance level has a power of 86%, 90%, and 95%, with effective sizes of 0.8, 0.90, and 1.00, respectively. To detect. For example, if the standard deviation of the log ratio is 2.0, a 5: 1 ratio in NM404 accumulation of tumor to normal tissue is detected with a power of 86%. Similarly, if the standard deviation of the log ratio is 1.6, a 5: 1 ratio of tumor to normal tissue accumulation of NM404 is detected with a power of 95%.

Determine whether NM404 can detect metastatic recurrence after first line treatment of prostate cancer (stage D0) in 10 patients with elevated PSA as the only evidence of disease

  Purpose and rationale

  By definition, patients with stage D0 prostate cancer have PSA elevation after the most reliable treatment of the disease, including conventional staging (CT and bone scans, and no radiographic evidence of disease Negative) and biochemical recurrence. Patients more likely to have a disease detectable by a NM404 PET-CT scan must be enrolled and consequently have an increase in PSA greater than 0.75 ng / ml / year.

Study Procedure: The imaging procedure in this study is identical to the original study, except that the patient is imaged only once based on the optimal imaging time identified in the initial protocol. Patients receive an injection of ¹²⁴ I-NM404 (0.3 μg / kg body weight, 1 mCi or limit established by dosimetry calculations in the first set of patients). The patient is also subjected to a PET-CT scan 48 hours after infusion. Vital signs are obtained after ¹²⁴ I-NM404 infusion.

  Statistical considerations: Ten patients with stage D0 prostate cancer are collected and followed for metastatic recurrence for at least 2 years. In this part of the study, all patients collected have PSA values greater than 0.75 ng / ml / year, so we expect at least 60% to experience metastatic recurrence within 2 years. Assuming that 6 out of 10 patients eventually experience metastatic recurrence within 2 years, the sensitivity of NM404 to detect metastatic recurrence is less than 20% standard error, and the 90% confidence interval for sensitivity is It is not wider than 55%. Moreover, the null hypothesis that the sensitivity of NM404 is at most 30% in this patient group is tested against the alternative hypothesis that the sensitivity is greater than 30%. The one-sample binomial test has a 83% power to detect 75% sensitivity, with a significance level of 10% on one side. A sensitivity of 80% is detected with a power of 90%.

  In the United States alone, approximately 230110 new prostate cancers are diagnosed in 2004 alone. Regardless of the technical improvement of the most reliable local treatment for prostate cancer, whose organs have been narrowed clinically by thorough prostatectomy, many men recover only with the primary treatment, and long-term follow-up As many as 40% of patients experience biochemical recurrence. The biggest challenge in treating patients with prostate cancer who have clinically narrowed organs or who have biochemical recurrence after receiving the most reliable treatment of a disease that is presumed to have narrowed organs One is still to accurately distinguish between local and metastatic disease. This diagnostic capability is important in identifying patients who can benefit from effective local treatment therapies, including surgery, external beam radiation, brachytherapy, and cryotherapy. Because we currently do not have a means of accurate staging, patients with metastatic disease that cannot be detected with the naked eye can receive unnecessarily local treatment at risk for treatment. Moreover, patients with elevated PSA due to local recurrence and who cannot confidently eliminate systemic recurrence can unnecessarily undergo hormone ablation. Hormone ablation is generally not thought to be effective for disease and is associated with progression of osteoporosis, loss of libido, weight gain, climacteric syndrome and overall discomfort, and progression of hormone-independent prostate cancer. ing.

  Conventional contrast studies (such as computed tomography (CT) and magnetic resonance imaging (MRI)) are useful for assessing soft tissue metastases, but most prostate cancers metastasize only to bone. Therefore, the use of CT and MRI scans is suboptimal in assessing disease, and there is a need for a more sensitive imaging treatment modality for either local recurrence or metastatic prostate cancer . Radioimmunoscintigraphy (ProstaScint, Cytogen Corp, Princeton, NJ) using indium-111 capromab pendide was performed in patients with post-prostatectomy with elevated PSA (in this patient, a clinical diagnosis of metastatic disease that cannot be detected with the naked eye). In other imaging studies, there is no clear evidence of metastatic disease). However, the use of ProstaScint scanning for patients at risk of metastasis from prostate cancer that cannot be detected with the naked eye still remains conventional. One approach to developing a sensitive imaging test is to develop a more appropriate carrier molecule that is key to achieving delivery of the radiopharmaceutical probe to the desired target tissue. One strategy is to study radioiodinated phospholipid ether analogs (PLE) as potential diagnostic contrast agents. Utilizing the inherent biochemical or pharmacological properties of these molecules results in a high degree of tissue or tumor selectivity. In preclinical models, the inventors have shown that these molecules are selectively accumulated at high levels in a wide variety of mouse and human tumors.

  Certain embodiments of the present invention provide preliminary data regarding the use of NM404, a second generation PLE analog, in imaging prostate cancer patients. NM404 is (a) highly selective retention in a wide variety of tumor types in preclinical models, (b) safe in humans, (c) radiolabeled with I-124 And (d) having appropriate dosimetric properties labeled with I-131.

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  All references cited herein are hereby incorporated in their entirety, in particular by reference and for the purpose of fully describing the present specification.

  The invention is not limited by the embodiments described by way of example in the specification, but encompasses all forms as set forth within the scope of the appended claims.

Brief Description of Drawings
Structure of specific PLE analogs Classic model showing the production of bremsstrahlung, characteristic X-rays, and Auger electrons. (Left) Electrons are scattered elastically and inelastically by positively charged nuclei. Inelastically scattered electrons lose energy and appear as bremsstrahlung. Elastically scattered electrons (including backscattered electrons) are generally scattered at a wider angle than inelastically scattered electrons. (Right figure) Incident electrons ionize sample atoms by emitting electrons from the inner shell (in this case, the K shell). De-excitation produces characteristic X-ray radiation (upper) or Auger electrons (lower) one after another. Secondary electrons are emitted with lower energy from the outer loosely bound electron shell (process not shown). Scintigraphy of the precordial area of patient 03 was obtained at 1, 2 and 6 days after intravenous administration of 1 mCi of 131I-NM-324. Uptake is seen in left tongue lung cancer (T) over time, with an increasing ratio of tumor to background. Time course (days) of NM404 in SCID mice bearing human RL-251 adrenal tumor (T) xenografts. Tissue distribution I-1125-NM404 in RL251 adrenal carcinoma in SCID mice, which showed a decrease in distribution in blood, spleen and kidney between days 1-14 with increasing tumor accumulation. Clear SCC1 and SCC6 tumor regression after injection of 125I-NM404. By day 41, tumors had decreased significantly. The image shows one of the tumor-bearing animals treated with I-125-NM404 (250 μCi) for 4 weeks after injection. The hair above the tumor has fallen off, presumably due to the accumulation of radioactivity intensity in the tumor. Moreover, due to bleeding and necrosis, the surface of the tumor appears to be “caved in”, indicating a darker area. The figure shows the effect of I-125-NM404 on tumors. Although the size of the tumor (outer dimension) cannot shrink, I-125-NM404 causes central necrosis. Measurement methods that measure tumor profile can underestimate the tumor volume response associated with I-125-NM404. PC3 human prostate cancer model implanted in SCID mice. PC3 is known to be radiation insensitive. The curve between control (no radioactivity, cold NM404) and treatment (I-125-NM404) only separates after about 4-5 weeks of treatment, until then the tumor growth is identical looks like. This suggests that: 1) NM404 takes several days to about a week to fully accumulate in the tumor, and 2) isotope I-125 has a low radiation flow. Have (because it has a long half-life). When all NM404 is cleared from normal tissue, both factors contribute to delaying the onset of therapeutic effect. NM404 (lower panel), NM324 (upper panel) and NM324 (upper) on days 1, 2, and 4 in SCID mice with human prostate PC-3 tumors (arrows) implanted in the flank Comparison with panel. Liver and background radioactivity is much improved with NM404. Tumor volumes for each group (controls and doses depicting 50 μCi, 150 μCi, 250 μCi and 500 μCi) were recorded over a 10 week evaluation period. In this figure, control animals show rapid tumor growth over a 10 week evaluation period. This ensures that the compound itself, C-NM404, has no substantial effect on tumor growth. The 50 μCi dose group does not show any difference from the control animals and therefore they are considered dose levels that are not effective in this animal model. However, the 150 μCi, 250 μCi and 500 μCi dose groups show a substantial and increased therapeutic effect. The tumor volume is constant and the same tumor appears to “collapse” (the surface of this tumor has collapsed). In addition, the hair over the tumor was lost, confirming the substantial accumulation of radioactivity in such a tumor. The results show the dose-linear effect of I-125-NM404 on tumor volume. A549 tumor xenografts (1 × 10 ⁶ cells, subcutaneous injection) in female SCID mouse divided doses (3 × 50 mCi). They have two independent controls for each dose. Divided doses of NM404 (eg, a single line dose of 3 × 50 mCi versus 150 mCi) produced the same therapeutic effect. The divided dose may be safer because the divided dose is drained from normal tissue within the time between divided injections. A549 large tumor vs small tumor (150 microcurie) Bioscan imaging (A) was obtained 4 days after ¹²⁵ I-NM404 injection into Apc ^Min mice. A digital photograph (B) of a mouse lung excised with a spontaneous lung tumor (diameter 2 mm, arrow) and a fused image (C), which is positionally matched, show strong uptake of NM404 in the tumor. Time course of ¹²⁵ I-NM404 in human RL-251 adrenal carcinoma xenografts in SCID mice. Residence with an enlarged tumor (1.5 x 0.5 cm, arrow) is evident even after 20 days. MRI imaging (blue) and 3D microPET imaging (A) imaging the surface of the fused 3D were obtained 24 hours after ¹²⁴ I-NM404 (80 μCi) intravenous injection into rats with CNS-1 glioma brain tumors. It was. The images were fused using Amira (v3.1). The lower panel shows (B) a coronary MRI section (MRI slice) that penetrated the tumor (arrow) and enhanced contrast, and (C) fused coronary MRI and ¹²⁴ demonstrating the presence and location of the tumor. 1 shows I-NM404 microPET imaging. The ¹²⁴ I- micro ^PET, for imaging the human A549 lung tumor xenografts in SCID mice injected 48 hours after 124 I-NM404 (80μCi). The corresponding image plane is indicated by a green dashed line in the coronal view. PC-3 (flank) ¹²⁴ I- micro PET imaging after 96 h of ¹²⁴ I-NM404 intravenous injection into tumor-bearing mice (coronal, sagittal and the shaft-like). Activity at the bladder was not evident at any time. ^{124 I-NM404 (80μCi) 124} I- micro PET imaging of transgenic mice with spontaneous c-myc pancreatic adenocarcinoma after injection of 18 hours (5 mm). Scintigraphy of NM404 (lower panel) and NM324 (upper panel) on days 1, 2, and 4 in SCID mice with human prostate PC-3 tumors (arrows) implanted in the flank comparison. Liver and background radioactivity is much improved with NM404. Time course of ¹²⁵ I-NM404 in human RL-251 adrenal carcinoma xenografts in SCID mice. Residence with an enlarged tumor (1.5 x 0.5 cm, arrow) is evident even after 20 days. A photograph of the resected prostate / vesicular gland complex (A). Bioscan image (B) obtained 4 days after NM404 injection. Fused photograph / Bioscan angiography (C) with the location of resected prostate / vesicular glands showing strong uptake of radioactivity in prostate tumors. High density 3D surface-rendered micro CT angiography (first) at various time points after intra-tibia injection of 2 × 10 ⁵ human PC3 prostate tumor cells (high density 3D surface-rendered) Panel shows injection site and direction). The tumor began to protrude through the bone by day 28 (arrow), and by day 46, the tumor literally destroyed the tibia, leaving only the ribs intact. Co-recorded surfaces were imaged, and micro-CT imaging of coronal sections fused with aligned Bioscan radionuclide scans 4 days after injection of ¹²⁵ I-labeled NM404 (color). Localized NM404 activity correlates well with the tibial PC3 tumor (arrow). MRI imaging (blue) and 3D microPET imaging (A) imaging fused 3D surfaces obtained 24 hours after intravenous injection of ¹²⁴ I-NM404 (100 μCi) to rats with CNS-1 glioma brain tumors . The images were fused using Amira (v3.1). The right panel shows (B) a coronary MRI section with enhanced contrast through the tumor (arrow), and (C) fused coronary MRI and ¹²⁴ I− confirming the presence and location of the tumor. NM404 microPET imaging is shown. 4 days after intravenous administration of ¹³¹ I-NM-404 (0.8 mCi) to patients with non-small cell lung cancer (diameter 6 cm, arrow), whole body planar planar nuclear imaging ) (A). Lung tumors are easily detected in corresponding axial computed tomography (CT) scans (B) and coronal computed tomography (CT) scans (C).

Claims (20)

In a subject with cancer or suspected of having cancer, lung cancer, adrenal cancer, melanoma, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma, intestinal cancer, Detect and locate recurrent, radiation and chemical insensitive or metastatic cancers selected from the group consisting of hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer and pancreatic cancer The method comprises the following steps:
Administering a phospholipid ether analog to the subject; and organs suspected of having recurred cancer, cancer insensitive to radiation or metastasized in the subject, rather than in the surrounding area (s) Determining whether to retain a high level of the analog, wherein the higher retention area indicates the detection and location of the cancer that has recurred, cancer that is insensitive to radiation and chemicals, or cancer that has metastasized. A method comprising the steps. 2. The method of claim 1, wherein the phospholipid analog is:

Wherein X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent, or

Where X is a radioisotope 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 , NH ₂ , NR ₂ and NR ₃ , wherein R is an alkyl or arylalkyl substituent,
More selected, the method. 3. The method of claim 2, wherein X is ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At. A method selected from the group of isotopes.   The method according to claim 2, wherein the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propane. Diol-3-phosphocholine, or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, wherein iodine is a radioisotope A method that is in form.   The method of claim 1, wherein the detection is performed by a PET, CT, MRI scanning method and combinations thereof. A method for the treatment of a relapsed cancer, a radiation and chemical insensitive cancer or a metastasized cancer in a subject comprising the following steps:
Administering to said subject an effective amount of a compound comprising a phospholipid ether analog.   The method according to claim 6, wherein the cancer that has recurred, cancer that is insensitive to radiation and chemicals, or cancer that has metastasized is lung cancer, adrenal cancer, melanoma, colon cancer, colorectal cancer, ovarian cancer, prostate A method that occurs in a group selected from cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma, intestinal cancer, hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer and carcinosarcoma. 7. The method of claim 6, wherein the phospholipid analog is:

X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ , where , R is an alkyl or arylalkyl substituent,
Or

X is a radioisotope of halogen; n is an integer between 8 and 30; Y is selected from the group consisting of H, OH, COOH, COOR and OR, Z is NH ₂ , Selected from the group consisting of NR ₂ and NR ₃ , wherein R is an alkyl or arylalkyl substituent;
More selected, the method. 9. The method of claim 8, wherein X is ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At and combinations thereof. A method selected from the group of radioactive halogen isotopes consisting of:   10. The method of claim 9, wherein an effective amount of the phospholipid ether analog is a combination of at least two isotopes, one having an orbital range of about 0.1 mm to 1 mm, and the other. Is a method wherein the trajectory range is about 1 mm to 1 m. 12. The method of claim 10, wherein the effective amount of the phospholipid ether analog is a combination of at least two isotopes, ¹²⁵ I and ¹³¹ I.   The method according to claim 6, wherein the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propane. Diol-3-phosphocholine, or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, where iodine is a radioisotope form Is that way.   7. The method of claim 6, wherein an effective amount of the phospholipid ether analog is resolved.   7. The method of claim 6, wherein an effective amount of the phospholipid ether analog is about 0.5 [mu] Ci to about 3 Ci and is treatable in a linear and dose-dependent manner.   15. The method of claim 14, wherein the dose is adjustable with respect to the volume of the cancer.   15. The method of claim 14, wherein the dose for the radiation and chemical insensitive tumor is greater than the dose for the radiation and chemical sensitive tumor and less than 3 Ci, A method that is adjustable relative to volume.   Use of a phospholipid ether analog for the manufacture of a pharmaceutical composition for the treatment of cancer that has recurred, cancer that is insensitive to radiation and chemicals, cancer that has metastasized. 18. The use according to claim 17, wherein the phospholipid analog is:

Where X is selected from the group consisting of radioisotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH ₂ , NR ₂ and NR ₃ Where R is an alkyl or arylalkyl substituent;
Or

Where X is a radioisotope 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 , NH ₂ , NR ₂ and NR ₃ , wherein R is selected from being an alkyl or arylalkyl substituent. X is selected from the group of radioactive halogen isotopes consisting of ¹⁸ F, ³⁶ Cl, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At and combinations thereof The use according to claim 18. 18. The use according to claim 17, wherein the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-O- [18- (p-iodophenyl) octadecyl] -1,3-propane. Diol-3-phosphocholine, or 1-O- [18- (p-iodophenyl) octadecyl] -2-O-methyl-rac-glycero-3-phosphocholine, where iodine is a radioisotope form Is used.

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