Methods for Imaging Integrin Expression and Endometriotic Sites

Abstract

The present invention relates to a radiopharmaceutical 99m The present invention relates to a method for imaging endometriosis sites using Tc-malaciclactide. The technetium-99m radiopharmaceutical is suitably prepared from a non-radioactive kit containing the RGD peptide malaciclactide. Also described is a method for diagnosing endometriosis, selecting treatment and monitoring treatment using the drug. The present invention also includes the use of the kit and / or gamma camera or gamma detector in the method of the present invention.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a radiopharmaceutical 99m The present invention relates to a method of imaging endometriosis sites using Tc-malaciclactide. The technetium-99m radiopharmaceutical is suitably prepared from a non-radioactive kit. Also described is a method of diagnosing endometriosis, selecting a treatment and monitoring the treatment using the drug. The present invention also includes the use of the kit and / or a gamma camera or gamma detector in the method of the present invention.

[0002] 2. Background of the Invention The endometrium is an epithelial lining that lies alongside the mucosa of the uterine cavity. The endometrium is normally shed once a month during menstruation and then regenerates. Endometriosis is a gynecological disorder of the female reproductive system, characterized by the presence of abnormal (i.e., ectopic, out-of-place) proliferative endometrial cells outside the uterus. The mechanism is not fully understood, but is thought to involve the attachment of parts of the endometrium shed during menstruation. Ectopic proliferation is commonly found within the pelvis, such as the ligaments supporting the ovaries and uterus. Less commonly, ectopic tissue may also attach to the outer surface of the small and large intestine, the ureters, bladder, vagina, or the lining of the abdominal and thoracic cavities. Thus, endometriosis may manifest as a superficial attachment to the peritoneum (a folded membrane that covers the digestive organs such as the intestine and pancreas); an ovarian cyst (endometrioma) or a deep infiltrating lesion typically located in the pelvic portion of the rectovaginal fascia, uterosacral ligaments, urinary or digestive tract. More rarely, endometriosis can occur outside the abdomen and pelvis, such as in the chest.

[0003] Although endometriosis is a benign disease, it can behave like a malignant disease by exhibiting abnormal proliferation and invasion of normal tissues. Endometriosis is estimated to affect 3-10% of the female population of reproductive age and is a debilitating disease that causes pelvic pain, sexual dysfunction, infertility, depression, and many other problems. In up to 20% of cases, a type of endometriosis called "deep infiltrating" or "deeply infiltrative endometriosis" (DIE) occurs deep within tissues and organs (i.e., at least 5 mm deep), especially in organs close to the uterus, including the bowel and bladder.

[0004] Endometriosis is difficult to diagnose, taking an average of 7 years from the onset of symptoms to definitive diagnosis. Clinical evaluation alone is unreliable for diagnosis. Current non-invasive techniques such as ultrasound (especially transvaginal ultrasound or TVS), MRI and blood tests such as serum CA-125 measurement have insufficient diagnostic accuracy. Ultrasound and MRI are useful in diagnosing ovarian endometrioma and DIE, respectively, but are of little or no value in diagnosing peritoneal endometriosis. Confirmation of the diagnosis therefore usually relies on characterization of the lesion by invasive laparoscopic surgery. Laparoscopy with lesion biopsy is the current "gold standard" diagnosis for endometriosis.

[0005] U.S. Patent No. 6,540,980 teaches a method for diagnosing endometriosis using an agent comprising a binding agent for eosinophil peroxidase linked to a detectable label, such as a radioactive label, an MRI agent or a fluorescent label.

[0006] US Pat. No. 9,861,711 teaches a method for the in vivo diagnosis of endometriosis using a labeled ligand of anti-Mullerian hormone.

[0007] Healy et al., Human Reprod. Update, 4 (5), 736-740 (1998) provide a review disclosing that endometrial angiogenesis is increased in women with endometriosis compared to controls. Healy et al. do not discuss imaging of endometriosis, but direct the study of agents that inhibit angiogenesis as a potential treatment for endometriosis.

[0008] Cosma et al. [J.Obstr.Gynaecol., 42(12), 1724-1733 (2016)] reported that in a PET / CT study of four DIE subjects, 18 studied the use of F-estradiol. They reported that it had better detection accuracy than MRI, but was unable to identify small nodules in the bladder wall. They suggested that PET / CT imaging should be performed during the first part of the patient's menstrual cycle, when estrogen expression is expected to be high and therefore the likelihood of visualization of endometriotic lesions is higher.

[0009] Fastrez et al. [Eur. J. Obst. Gynecol. Reprod. Biol., 212, 69-74 (2017)] tested a PET radiotracer agent targeting somatostatin receptors in a pilot clinical trial in patients with endometriosis. 68 disclosed the use of Ga-labeled dotatate. They reported low sensitivity and specificity in diagnosing rectovaginal lesions of DIE.

[0010] Silveira et al. [Reprod.Sci., 25(1), 19-25 (2018)] reported the use of radioactive tracers in a rat model of endometriosis. 18 disclose the use of F-fluorocholine imaging. They suggest that this technique should be further evaluated for the detection of small superficial endometriotic lesions in humans.

[0011] Amartuvshin et al. [J.Endomet.Pelv.Pain Dis., 11(4), 194-200 (2019)] disclose imaging studies in a mouse model of endometriosis. They argue that vascular endothelial growth factor (VEGF) is essential for the survival and growth of endometriotic cells and therefore should be used in combination with PET radiotracers. 64 used the Cu-labeled antibody bevacizumab (an anti-VEGF antibody). They concluded that this tracer may be useful for imaging endometriosis, but no human studies have been reported since.

[0012] US Pat. No. 10,259,844 teaches a method for imaging endometriosis based on the peptide Val-Arg-Arg-Ala-Asp-Asn-Arg-Pro-Gly labeled with a detectable marker.

[0013] WO2005 / 030265 teaches an imaging agent for optical imaging of endometriosis, which comprises a vector of abnormally expressed biological targets associated with endometriosis conjugated to an optical reporter moiety.A wide variety of such biological targets are described, including angiogenesis targets; progesterone receptors; estrogen receptors and folate binding proteins.However, WO2005 / 030265 does not provide data on the efficacy or otherwise of any of the possible biological approaches described therein.

[0014] Velikyan et al. [Am. J. Nucl. Med. Mol. Imaging, 8(1), 15-31 (2018)] described a method for noninvasive imaging of angiogenesis. 68 Methods for the synthesis of Ga-labeled RGD peptides are taught. Velikyan et al. prepared chelator conjugates of the bicyclic RGD octapeptide NC100717, the chelator being DOTA. They report specific uptake into the uterine wall in non-human primate imaging studies. They note that angiogenesis is involved in cancer, connective diseases, psoriasis, arthritis, endometriosis, multiple sclerosis, and obesity.

[0015] WO 03 / 006491 describes a compound of formula (I): [ka] [In the formula, G represents glycine, D represents aspartic acid, R 1 is -(CH 2 ) n -or-(CH 2 ) n -C 6 H 4 -, where n is a positive integer from 1 to 10; h represents a positive integer 1 or 2; X 1 represents an amino acid residue, where said amino acid has a functional side chain, such as an acid or amine; X 2 and X 4 represents an amino acid residue capable of independently forming a disulfide bond, X 3 represents arginine, N-methylarginine or an arginine mimetic, X 5 represents a hydrophobic amino acid or a derivative thereof, X 6 represents a thiol-containing amino acid residue, X 7 represents either absent or a biomodifying moiety, Z 1 represents an antitumor agent, a chelator, or a reporter moiety, and W 1 is absent or represents a spacer moiety, or a pharma- ceutically acceptable salt thereof.

[0016] WO 03 / 006491 describes preferred chelating moieties as having the formula shown below: [ka] wherein said chelator conjugate of formula I 99m It has been disclosed that it contains a Tc complex.

[0017] WO 03 / 006491 states that "diseases and indications associated with angiogenesis are, for example, different forms of cancer and metastasis, such as breast cancer, skin cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer or ovarian cancer. Other diseases and indications are inflammation (for example chronic), atherosclerosis, rheumatoid arthritis and gingivitis. Other diseases and indications associated with angiogenesis are arteriovenous malformations, astrocytoma, choriocarcinoma, glioblastoma, glioma, hemangioma (childhood, capillary), hepatocellular carcinoma, endometrial hyperplasia, ischemic myocardium, endometriosis, Kaposi's sarcoma, macular degeneration, melanoma, neuroblastoma, occlusive peripheral arterial disease, osteoarthritis, psoriasis, retinopathy (diabetic, proliferative), scleroderma, seminoma and ulcerative colitis." However, this is an extensive list of possibilities and WO 03 / 006491 does not disclose which agents and "reporter moieties" within this range would be useful for what disease states, or how each of these disease states might be imaged in a usefully interpretable way.

[0018] In the specification of European Patent No. 2598175, 99m Radiopharmaceutical compositions of the bicyclic RGD peptide malaciclatide labeled with Tc and stabilized with para-aminobenzoic acid (pABA) are disclosed, as well as non-radioactive kits containing pABA for preparing such radiopharmaceutical compositions.

[0019] U.S. Patent No. 10,729,793 discloses a method for imaging arthritis using radiopharmaceuticals based on chelator conjugates of bicyclic RGD peptides similar to those of formula I of WO 03 / 006491 (noted above). Preferred such radiopharmaceuticals for imaging arthritis include: 99m Tc-malaciclactide.

[0020] SUMMARY OF THE PRESENT APPLICATION There is a need for non-invasive imaging methods useful in diagnosing endometriosis. The present invention provides a non-invasive radiopharmaceutical imaging method to aid in the diagnosis of endometriosis. The method avoids the routine need for surgical intervention (such as laparoscopy). Also, 18 F or 68 The radiation dose to the patient is lower than that of conventional PET agents that use positron-emitting radioisotopes such as Ga. 99m Tc-malaciclactide is rapidly cleared in vivo, which helps reduce radiation dose to the patient and is advantageous for imaging. 99m A generator-produced radioisotope along with a non-radioactive kit for the preparation of Tc-malaciclactide 99m Because it uses Tc, the radiopharmaceutical itself is expected to be widely available. 99m Because Tc is a gamma-ray emitting radioisotope that has long been established in nuclear medicine, there is a large installed base of gamma cameras suitable for the imaging of the present invention. 99m The use of Tc radionuclides allows for wider access to diagnosis and significantly shortens the mean time from symptom onset to final diagnosis, allowing for earlier treatment and therefore reducing the need for laparoscopic surgery. The method also helps to identify women who do not have active endometriosis and therefore individuals who should pursue alternative diagnoses.

[0021] In descending order of frequency, endometriosis appears as superficial peritoneal lesions, endometriomas, deep infiltrating endometriosis (DIE) and extrapelvic lesions. Unlike some prior art methodologies, the present method is applicable to superficial endometriotic lesions, i.e., not limited to deep infiltrating endometriosis (DIE). Thus, only ovarian endometriomas and deep nodular forms of the disease can be detected by ultrasound and MRI. Also, ultrasound is time-consuming, operator-dependent, often uncomfortable or invasive (especially when performed transvaginally, as in endometriosis), and cannot detect the most common forms of the disease. MRI is time-consuming, expensive, has a very limited ROI (region of interest) or geographic focus on the patient, and also cannot detect the most common forms of the disease. Thus, neither offers the possibility of whole-body imaging, nor imaging of the subject's entire abdomen and pelvis, nor can it detect the majority of cases. The present method is capable of rapidly imaging all areas related to endometriosis (pelvis, abdomen and chest) within one imaging session without causing discomfort to the patient.

[0022] The method is also applicable to peritoneal endometriosis, where ultrasound and MRI have little diagnostic value. As the method is not estradiol-based, it can be performed at any phase of the menstrual cycle. Furthermore, the lesions can be visualized radiographically, allowing for the monitoring of endometriosis treatment and the testing of potential new treatments for this disease.

[0023] Detailed Description of the Invention In a first aspect, the present invention provides a method of imaging sites of endometriosis in a subject, comprising administering a radiopharmaceutical 99m The subject is pre-administered with Tc-malaciclactide, followed by 99m and in vivo ex utero imaging of radioactive emissions from Tc-malaciclactide, said site being: (i) Abdominal endometriosis; (ii) peritoneal endometriosis; (iii) Thoracic endometriosis The present invention provides a method comprising:

[0024] The terms "comprising" or "comprises" have their conventional meaning throughout this application, meaning that the composition must have the recited components, but that other unspecified compounds or species may additionally be present. The term "comprising" includes "consisting essentially of" as a preferred subset, which means that the composition has the recited components, without other compounds or species being present.

[0025] The term "subject" refers to an in vivo intact mammalian body, preferably a human female patient, more preferably a human female of reproductive age, for the latter, the present invention is expected to be most useful during the proliferative phase of the menstrual cycle when the endometrium grows most rapidly in preparation for the potential implantation of a fertilized egg.

[0026] 99m Tc is the radioisotope technetium-99m, which decays to technetium-99( 99 Radioactive decay is accompanied by the emission of gamma rays with a photon energy of 140 keV, which is nearly ideal for medical imaging. These gamma rays are the first mode of "radioactive emission." 99m Tc and 99m Tc-radiopharmaceuticals are well known in the art and include 99m Tc is readily available from commercial technetium-99m generators. 99m Tc is also used in cyclotrons. 99 Mo or 100 It can also be produced by irradiating Mo. 99m For information on Tc radiopharmaceuticals, imaging methods, and related kits, see Technetium-99m Pharmaceuticals: Preparation and Quality Control in Nuclear Medicine, I. Zolle (Ed), Springer (2006).

[0027] Malaciclatide is the recommended INN (United States Approved Name) of NC100692. The term "malaciclatide" refers to the compound known in the scientific literature as NC100692 [D. Edwards et al., Nucl. Med. Biol., 35, 365-375 (2008)]. The chemical structure of malaciclatide is as follows: [ka]

[0028] Malaciclatide is a bicyclic RGD (Arg-Gly-Asp) peptide conjugated to a diaminedioxime chelator that forms a metal complex with the technetium radiometal, which is a neutral complex.

[0029] Malaciclazide may be used in the form of a free base or in the form of a salt (e.g., a trifluoroacetate salt). 99m Synthesis of Tc-malaciclactide, and 99m A kit for preparing Tc-malaciclactide is presented in this example.

[0030] The term "radiopharmaceutical" has its conventional meaning and refers to a radioactive compound in a form suitable for in vivo mammalian administration for use in diagnosis or therapy. The phrase "in a form suitable for mammalian administration" refers to a composition that is sterile, pyrogen-free, free of compounds that produce toxic or deleterious effects, and formulated at a biocompatible pH (for the agents of the invention, about pH 4.0-10.5, preferably pH 6.5-9.5) and a physiologically compatible osmolality. Such compositions are free of particulates that may pose a risk of causing embolism in vivo and are formulated so as not to precipitate on contact with biological fluids (e.g., blood). Such compositions also contain only biologically compatible excipients and are preferably isotonic.

[0031] The present invention 99m The Tc-malaciclactide radiopharmaceutical is provided in a biocompatible carrier. A "biocompatible carrier" is a fluid, particularly a liquid, in which the radiopharmaceutical can be suspended or preferably dissolved so that the composition can be administered to the mammalian body in a physiologically acceptable manner, i.e., without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid, such as sterile pyrogen-free water for injection; an aqueous solution, such as saline (which can be advantageously equilibrated so that the final product for injection is isotonic); an aqueous buffer solution containing a biocompatible buffering agent (e.g., phosphate buffer); an aqueous solution of one or more tonicity adjusters (e.g., salts of plasma cations and biocompatible counterions), sugars (e.g., glucose or sucrose), sugar alcohols (e.g., sorbitol or mannitol), glycols (e.g., glycerol), or other non-ionic polyol materials (e.g., polyethylene glycol, propylene glycol, etc.). Preferably, the biocompatible carrier is pyrogen-free water for injection, isotonic saline or phosphate buffer.

[0032] The radiopharmaceutical composition is suitably provided in a pharmaceutical grade container. A preferred such container is a septum-sealed vial, in which an airtight closure is crimped with an overseal (usually of aluminium). The closure is suitable for single or multiple punctures by a hypodermic needle (e.g. a crimp-on septum-sealed closure) whilst maintaining sterile integrity. Such containers have the added advantage that the closure can withstand vacuum if required (e.g. for changing headspace gas or degassing the solution) and can withstand pressure changes such as reduced pressure without allowing ingress of external atmospheric gases such as oxygen or water vapour. A preferred multi-dose container is a single bulk vial (e.g. 6-30 cm2) containing multiple patient doses. 3 volume) so that single patient doses can be withdrawn into clinical grade syringes at various time intervals during the life of the formulation to suit the clinical situation.

[0033] The radiopharmaceutical composition may also be provided in a syringe, the pre-filled syringe being designed to contain a single human dose, i.e. a "unit dose", and is therefore preferably a syringe suitable for clinical use, such as single use.

[0034] The term "pre-administration" refers to administration of a first dose of a therapeutic agent before the imaging of the first aspect is performed. 99m This refers to the fact that a Tc-malaciclactide radiopharmaceutical is administered to a subject. 99m The Tc-malaciclactide radiopharmaceutical is administered to a subject in a minimally invasive manner, i.e., without substantial health risk to the mammalian subject, even when performed under specialized medical expertise. Such minimally invasive administration is preferably intravenous administration into a peripheral vein of the subject, without the need for local or general anesthesia.

[0035] The phrase "one or more" in options (i)-(iii) means that these may be imaged alone, or a combination of two of them, or all three, by judicious selection of the detector / camera field of view and / or region of interest (ROI), or by torso or whole body imaging of the subject.

[0036] The term "extrauterine" refers to all parts of the body outside the uterine body and endometrium. The term "endometrium" refers to the inner epithelial layer of the uterus and its mucosa. Screening for extrauterine endometriosis therefore refers to radiological visualization of ectopic endometrial material.

[0037] The term "abdominal endometriosis" refers to the conventional meaning, i.e., the abdominal region of a subject, with the area including the stomach, liver, kidneys and intestines of the subject. The term "pelvis" refers to the body part immediately below the abdomen that forms the lower back of the torso and includes the pelvis, pelvic floor and pelvic cavity, and perineum. In female subjects of the present invention, this includes the ligaments that support the ovaries and uterus. The term "pelvis" has its conventional meaning, and thus "pelvic endometriosis" refers to the pelvic region of a subject.

[0038] The term "peritoneal endometriosis" refers to endometriosis that is part of the peritoneal anatomical structures, particularly its contents (i.e., the small intestine, large intestine and pancreas), or the peritoneum itself that covers these organs.

[0039] The term "superficial endometriosis lesion" refers to endometrial tissue attached to the surface of an organ or anatomical structure, as opposed to the DIE definition of having a penetration depth of more than 5 mm.The term "thoracic endometriosis" refers to endometrial tissue in the chest of a subject.Thus, thoracic endometriosis includes not only lung, but also lesions that are outside the lung but inside the chest, i.e., inside the thoracic cavity.

[0040] In the method of the first aspect, the superficial endometriotic lesion is preferably imaged in combination with abdominal, peritoneal or thoracic endometriosis.The method of the first aspect is most preferably used for imaging the superficial endometriotic lesion in combination with peritoneal endometriosis, i.e. for imaging the superficial peritoneal disease.

[0041] In the method of the first aspect, all three of (i) to (iii) may be imaged together, preferably via whole-body imaging of the subject. The term "whole-body imaging" refers to imaging of the entire body of a subject in a single imaging procedure.

[0042] In the method of the first aspect, the radioactive emission is preferably detected and processed using a conventional or high-resolution gamma camera; a gamma detector; image processing software or a combination thereof. A conventional gamma camera or "Anger camera" usually comprises a sodium iodide scintillator crystal coupled to an array of photomultiplier tubes. In the present invention, the gamma camera is preferably a high-resolution gamma camera capable of 360-degree imaging and has sub-millimeter spatial resolution. The gamma camera is most preferably a high-resolution gamma camera comprising a CZT detector. A "CZT detector" is a Cd-Zn-Te or cadmium-zinc-telluride solid-state detector, and suitable such cameras with CZT detectors are commercially available as Veriton CT, D-SPECT (Spectrum Dynamics Medical, Israel) or Starguide / Discovery NM / CT 870 CZT, NM 530c (GE Healthcare). Imaging typically begins 1 min to 36 h after administration of the radiopharmaceutical of the invention, with image acquisition by planar scintigraphy, SPECT or SPECT-CT for 5 to 45 min. Further information regarding radiopharmaceutical emission detection for medical imaging and / or diagnosis is provided in the second aspect (below). The sensitivity of CZT detectors allows for effective imaging with smaller radiopharmaceutical dosages, thus reducing the radiation burden to the subject. This also allows for, for example, background clearance from organs that may adversely affect the signal-to-background ratio. 99m This allows for effective imaging at longer times after administration, when it may be necessary to wait for several half-lives of Tc. CZT detectors also provide improved resolution over conventional SPECT imaging.

[0043] Some prior art radiotracer imaging agents suffer from background problems. 18 F-estradiol accumulates in the abdominal region, 18 F-choline accumulates heavily in the urinary tract. Both present significant drawbacks in terms of signal to background ratio when attempting to image the pelvic region.99m Tc-malaciclactide shows some background uptake in the bladder and intestine. There is background activity in the peritoneal region, but this can be distinguished by the high level of signal, i.e., signal-to-background ratio, in endometriotic lesions. Endometriotic lesions outside the abdomen (e.g., lungs), although rare, 99m It is easily visible due to the low background of Tc-malaciclactide.

[0044] In the method of the first aspect, 99m The Tc-malaciclazide radiopharmaceutical may be prepared by reacting malaciclazide in the presence of a reducing agent, as is known in the art for technetium radiopharmaceuticals. 99m It is suitably prepared by reaction with Tc-pertechnetate. 99m The Tc-malaciclazide radiopharmaceutical is preferably prepared by dissolving in a lyophilized non-radioactive kit containing malaciclazide. 99m Such kits are preferably prepared by reconstituting the kit with a solution of Tc-pertechnetate. a) Malaciclactide; b) para-aminobenzoic acid or a salt thereof with a biocompatible cation; c) stannous reducing agents; d) methylenediphosphonic acid or a salt thereof with a biocompatible cation Includes.

[0045] Para-aminobenzoic acid is commercially available, including in pharmaceutical grade purity. Preferably, pharmaceutical grade material is used. The term "biocompatible cation" refers to a positively charged counterion that forms a salt with an ionized negatively charged group, said positively charged counterion also being non-toxic and therefore suitable for administration to the mammalian body, particularly the human body. Examples of suitable biocompatible cations include the alkali metals sodium or potassium; the alkaline earth metals calcium and magnesium; and ammonium ions. The preferred biocompatible cations are sodium and potassium, most preferably sodium. Most preferably, the para-aminobenzoate salt of the present invention consists essentially of sodium para-aminobenzoate.

[0046] The term "kit" refers to one or more pharmaceutical grade containers containing the non-radioactive chemicals necessary to prepare a desired radiopharmaceutical composition, together with instructions for use. 99m Tc, particularly pertechnetate, is designed to be reconstituted with minimal manipulation to provide a solution suitable for administration to humans. The kit of the present invention preferably comprises a lyophilized composition containing all of the kit components in a single lyophilized formulation in a single container.

[0047] The term "stannous reducing agent" refers to 99m Tc has its traditional meaning in the field of radiopharmaceuticals and kits, and Sn 2+ i.e., stannous in the Sn(II) oxidation state. Such suitable salts may be in hydrated or anhydrous form and include stannous chloride, stannous fluoride and stannous tartrate. A preferred such stannous reducing agent is stannous chloride. The term "methylene diphosphonic acid" has its conventional chemical meaning and is abbreviated as MDP.

[0048] The kit is preferably lyophilized, 99m Sterile from a Tc radioisotope generator 99m Tc-pertechnetate (TcO 4 -) to provide a solution suitable for human administration without further manipulation. The kit preferably further comprises a buffer comprising a mixture of sodium bicarbonate and anhydrous sodium carbonate.

[0049] The most preferred kit formulation is as follows (Example 7): [Table 1]

[0050] The imaging method of the first embodiment is useful for diagnosing endometriosis, but is not completely diagnostic, since the radiopharmaceutical images obtained require one or more of the following: (a) comparison with normal, i.e. disease-free images; (b) interpretation by a clinical expert; or (c) analysis by a suitable computerized algorithm or artificial intelligence (alone or in combination). This diagnostic method is described in the second embodiment below.

[0051] In a second aspect, the present invention provides a method for diagnosing endometriosis comprising the imaging method defined in the first aspect.

[0052] In a second aspect, the site of endometriosis, the subject, and detection of radioactive emissions; 99m Tc-malaciclactide radiopharmaceutical; 99m Preferred embodiments of the method for preparing Tc-malaciclactide and the non-radioactive kit are as described in the first embodiment (above).

[0053] The diagnostic method of the second aspect includes: 99m The method includes administering a Tc-malaciclactide radiopharmaceutical to a subject.

[0054] The imaging of the first embodiment provides a medical image. Data from imaging of a subject using a suitable gamma camera or gamma detector is processed by algorithms known in the art to generate a medical image. Gamma cameras, gamma detectors, and image processing are reviewed in: Handbook of Nuclear Medicine and Molecular Imaging for Physicists, M. Ljungberg (Ed), 3-volume set, CRC Press (2021). And image interpretation for a given subject is accomplished by options A and B.

[0055] Option A (i) Review of images by a radiologist; (ii) qualitative or quantitative scoring of radiopharmaceutical uptake in regions of interest (ROIs); (iii) the decision by the radiologist or clinical team to proceed to laparoscopic surgery, to begin a course of treatment, or to exclude endometriosis as a diagnosis and pursue alternative approaches.

[0056] Option B (i) image analysis using one or more machine learning derived algorithms to highlight lesions within the ROI; (ii) review of images by a radiologist; (iii) the decision by the radiologist or clinical team to proceed to laparoscopic surgery, to begin a course of treatment, or to exclude endometriosis as a diagnosis and pursue alternative approaches.

[0057] In both options A and B, the radiologist may optionally refer to one or more normal scans. The term "normal scan" refers to at least one reference image using the same radiopharmaceutical, preferably the same gamma camera, detection means and image processing method, in another subject in which endometriosis was not found. Such two or more normal scans can be used as a library of images to assist in the normal variation of images. Thus, when laparoscopic surgery is utilized, a definitive diagnosis can be obtained, especially when used in conjunction with a biopsy that allows clinical characterization of the lesion, to help determine the subsequent management or treatment of the patient.

[0058] In a third aspect, the present invention provides a method of determining treatment for endometriosis in a subject comprising the imaging method of the first aspect or the diagnostic method of the second aspect.

[0059] In a third aspect, the site of endometriosis, the subject, and detection of radioactive emissions; 99m Tc-malaciclactide radiopharmaceutical, 99m Preferred embodiments of the preparation method and non-radioactive kit of Tc-malaciclactide are as described in the first embodiment (above). Preferred embodiments of the diagnostic method in the third embodiment are as described in the second embodiment.

[0060] In this embodiment, the imaging of the first embodiment can confirm the location of the endometrial tissue. That in itself may be sufficient information to help determine subsequent patient management or treatment. In other situations, the diagnostic method of the second embodiment is necessary. Knowledge of the disease location may help determine subsequent treatment or therapy (either surgery or drug therapy or a combination thereof). Treatment of endometriosis has been reviewed by KT Zondervan et al. [New Eng.J.Med., 383, 1246-1256 (2020)].

[0061] In a fourth aspect, the present invention provides a method of monitoring the treatment of endometriosis in a subject comprising the imaging method of the first aspect or the diagnostic method of the second aspect.

[0062] A fourth aspect of the present invention relates to a site of endometriosis, a subject, and detection of radioactive emissions. 99m Preferred embodiments of the preparation method and non-radioactive kit of Tc-malaciclactide are as described in the first embodiment (above). Preferred embodiments of the diagnostic method in the fourth embodiment are as described in the second embodiment (above).

[0063] The term "monitoring" refers to performing multiple imaging of the same subject before a course of treatment and at selected time intervals during the course of treatment. Comparison of these images can provide the clinician with information on whether the treatment has reduced the extent of endometriosis in the subject's ROI, or whether the attempted treatment has proven ineffective by progressing existing lesions or generating new lesions. It is therefore expected that the informational or diagnostic method of the present invention will help determine whether a given course of treatment has been successful for an individual subject. If a positive result is found, subsequent imaging can be used to confirm the progress of the treatment. If negative, an alternative therapy can be selected and new monitoring according to this fourth aspect of the present invention can be initiated.

[0064] In a fifth aspect, the present invention provides a method for the preparation of a medicament for the treatment of a cancer, comprising the steps of: (i) the imaging method of the first aspect; (ii) a diagnostic method according to the second aspect; (iii) a method of determining treatment according to the third aspect; (iv) a method of monitoring therapy according to the fourth aspect; For use in 99m There is provided a non-radioactive kit for the preparation of Tc-malaciclactide, said kit being as defined in the first aspect.

[0065] This fifth aspect also includes the use of the non-radioactive kit described above in one or more of the methods (i) to (iv).

[0066] A fifth aspect of the present invention relates to a site of endometriosis, a subject, and detection of radioactive emissions. 99m Preferred embodiments of the method for preparing Tc-malaciclactide and the non-radioactive kit are as described in the first embodiment (above).

[0067] In a fifth aspect, the kit comprises: 99m The non-radioactive kit is then used in a method for preparing the Tc-malaciclactide radiopharmaceutical, which is then used in one or more of methods (i) to (iv). Thus, the non-radioactive kit can be used to prepare malaciclactide and / or 99m Any of methods (i)-(iv) that provide Tc-malaciclactide are within the scope of this embodiment.

[0068] In a sixth aspect, the present invention provides a method for the preparation of a medicament for the treatment of a cancer, comprising the steps of: (i) the imaging method of the first aspect; (ii) a diagnostic method according to the second aspect; (iii) a method of determining treatment according to the third aspect; (iv) a method of monitoring therapy according to the fourth aspect; The present invention provides a use of a gamma camera, a gamma detector and / or image processing software in a method for detecting a gamma wave.

[0069] A sixth aspect of the present invention relates to a site of endometriosis, a subject, and detection of radioactive emissions. 99m Preferred embodiments of the method for preparing Tc-malaciclactide and the non-radioactive kit are as described in the first embodiment (above).

[0070] In a sixth aspect, the phrase "image processing software" refers to algorithms, in particular artificial intelligence algorithms, in particular 99m The gamma camera, gamma detector, and such image processing are described in the Handbook of Nuclear Medicine and Molecular Imaging for Physicists, It is reviewed in M. Ljungberg (Ed), 3-volume set, CRC Press (2021). Artificial intelligence in medical imaging is reviewed by Seah et al. [Br.J.Radiol., 94(1126), 20210406 ​​(2021)]. Machine learning in medical imaging is described by T. Sadad et al. [Curr.Med.Imaging, 17(6), 686-694 (2021)].

[0071] In a further aspect, the present invention provides a radiopharmaceutical for use in an in vivo imaging method for diagnosing extrauterine endometriosis in a subject. 99m The present invention provides Tc-malaciclactide, which is an endometriosis treatment for the following endometriosis: (i) Abdominal endometriosis; (ii) peritoneal endometriosis; (iii) Thoracic endometriosis The nucleic acid sequence may include one or a combination of the above sites.

[0072] In this further aspect, the definitions of the terms and preferred embodiments thereof are as described in the first and second aspects (above).

[0073] The invention is illustrated by the non-limiting examples detailed below. Examples 1-3 provide the synthesis of chelator 1 of the invention (sometimes referred to as carba-Pn216). Example 4 provides the synthesis of chelator 1A of the invention (an active ester functionalized version of chelator 1). Example 5 provides the synthesis of cyclic peptides of the invention and conjugation of chelators. Example 6 provides the synthesis of malaciclactide. Example 7 provides the preparation of a malaciclactide lyophilization kit. Example 8 provides the synthesis of chelator 1A of the invention (an active ester functionalized version of chelator 1). Example 9 provides the synthesis of cyclic peptides of the invention and conjugation of chelators. Example 10 provides the synthesis of malaciclactide. Example 11 provides the synthesis of malaciclactide lyophilization kit. Example 12 provides the synthesis of malaciclactide lyophilization kit. Example 13 provides the synthesis of malaciclactide lyophilization kit. Example 14 provides the synthesis of malaciclactide lyophilization kit. Example 15 provides the synthesis of malaciclactide lyophilization kit. 99m A method for reconstituting the kit to obtain the Tc-malaciclactide radiopharmaceutical is provided. Example 9 provides: 99m Tc-malaciclactide uptake and retention was demonstrated in three experimental models, suggesting that it may be beneficial for endometriotic tissue uptake. 99mWe provide biological data supporting the usefulness of Tc-malaciclactide. [Brief description of the drawings]

[0074] [Figure 1] Figure 1 compares the uptake of 99mTc-malaciclactide and the scrambled control peptide 99mTc-NC100667 in a rat model of endometriosis. Data are expressed as %id / g and relative concentrations and shown as mean ± SD; *=p<0.05 endometrial lesions 2 hours after injection. Figure 1 shows that 99mTc-malaciclactide (expressed as %id / g) showed 7-fold higher uptake and retention in endometrial lesions compared to the scrambled control peptide 99mTc-NC100667. This indicates that uptake into the endometrium was specific, as it requires the specific chemical structure of 99mTc-malaciclactide.

[0075] [Diagram 2] Figure 2 shows the uptake of 99mTc-malaciclactide in the same endometrial rat model. Angiogenesis of endometrial lesions was measured by microvessel density (MVD). Data are presented as mean (a-MVD) and highest value (h-MVD) and shown as mean ± SD; *=p<0.05. Uptake in endometrial lesions was associated with the level of angiogenesis (expressed as MVD) and was higher compared to control lesions in abdominal fat. This indicates that uptake of 99mTc-malaciclactide is specific to endometrial tissue.

[0076] [Diagram 3]Figure 3 shows the uptake of 99mTc-malaciclactide into isogenic mouse endometrial lesions 1, 2 or 3 weeks after implantation (2 hours after injection). Data are expressed as %id / g and shown as mean ± SD; *=p<0.05. Time course experiments were performed using the isogenic mouse model 1, 2 or 3 weeks after uterine horn inoculation. Uptake of 99mTc-malaciclactide was maximal 1 week after uterine horn inoculation. Therefore, subsequent biodistribution studies examined uptake of 99mTc-malaciclactide at the 1 week time point.

[0077] [Figure 4] Figure 4 shows that the initial uptake of 99mTc-malaciclactide in the lesions was 5.6%id / g 5 min after injection and decreased over time to 3.6%id / g, with 62% of the initial uptake of 99mTc-malaciclactide retained at 120 min after injection. In comparison, retention in the ovaries and uterine horns was approximately 35%. This suggests that 99mTc-malaciclactide was specifically retained in the lesions to a greater extent than in normal tissues. Furthermore, the ratios of endometrial lesions to blood and muscle reached 4.0 and 8.3, respectively, at 120 min.

[0078] Abbreviation Conventional one-letter or three-letter amino acid abbreviations are used. Ac: Acetyl Boc: tert-butyloxycarbonyl tBu: tert-butyl DIE: Deeply infiltrating endometriosis DMF: Dimethylformamide DMSO: Dimethyl sulfoxide Fmoc: 9-fluorenylmethoxycarbonyl HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU: O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate HPLC: High-performance liquid chromatography MDP: methylene diphosphonic acid MRI: Magnetic resonance imaging NMM: N-methylmorpholine pABA: para-amino-benzoic acid sodium salt PBS: Phosphate-buffered saline PEG: Polyethylene glycol, (OCH 2 CH 2 ) n (wherein n is an integer) PET: Positron Emission Tomography RCP: Radiochemical purity. ROI: Region of Interest RP-HPLC: Reversed-phase HPLC SPECT: Single-photon emission computed tomography TFA: Trifluoroacetic acid THF: tetrahydrofuran TIS: Triisopropylsilane TLC: Thin Layer Chromatography Trt: Trityl [ka]

[0079] Example 1: Synthesis of 1,1,1-tris(2-aminoethyl)methane. Step 1(a): 3(Methoxycarbonylmethylene)glutaric acid dimethyl ester. Carbomethoxymethylenetriphenylphosphorane (167 g, 0.5 mol) in toluene (600 ml) was treated with dimethyl 3-oxoglutarate (87 g, 0.5 mol) and the reaction was heated to 100 °C in a 120 °C oil bath under nitrogen for 36 h. The reaction was then concentrated under vacuum and the oily residue was triturated with 40 / 60 petroleum ether / diethyl ether (1:1, 600 ml). Triphenylphosphine oxide precipitated and the supernatant was decanted / filtered. The residue evaporated under vacuum was Kugelrohr distilled under high vacuum Bpt (oven temperature 180-200 °C, 0.2 torr) to give 3-(methoxycarbonylmethylene)glutaric acid dimethyl ester (89.08 g, 53%). [Table 2]

[0080] Step 1(b): Hydrogenation of 3-(methoxycarbonylmethylene)glutaric acid dimethyl ester. 3-(Methoxycarbonylmethylene)glutaric acid dimethyl ester (89 g, 267 mmol) in methanol (200 ml) was shaken with (10% palladium on charcoal:50% water) (9 g) under an atmosphere of hydrogen gas (3.5 bar) for 30 hours. The solution was filtered through diatomaceous earth and concentrated in vacuo to give 3-(methoxycarbonylmethyl)glutaric acid dimethyl ester as an oil (yield 84.9 g, 94%). [Table 3]

[0081] Step 1(c): Reduction of the trimethyl ester to the triacetate and esterification. Lithium aluminum hydride (20 g, 588 mmol) in THF (400 ml) was carefully treated with tris(methyloxycarbonylmethyl)methane (40 g, 212 mmol) in THF (200 ml) in a three-necked 2 L round bottom flask under nitrogen atmosphere over 1 hour. A strong exothermic reaction occurred, causing the solvent to reflux strongly. The reaction was heated to reflux in a 90° C. oil bath for 3 days. The reaction was quenched by careful dropwise addition of acetic acid (100 ml) until hydrogen evolution ceased. The stirred reaction mixture was carefully treated with acetic anhydride solution (500 ml) at a rate that gave a gentle reflux. The flask was equipped for distillation and stirred, then heated at 90° C. (oil bath temperature) to distill off the THF. Further acetic anhydride (300 ml) was added and the reaction was returned to reflux configuration and stirred and heated in an oil bath at 140° C. for 5 hours. The reaction was cooled and filtered. The aluminum oxide precipitate was washed with ethyl acetate and the combined filtrates were concentrated under vacuum (5 mmHg) on ​​a rotary evaporator at 50°C water bath temperature to give an oil. The oil was taken up in ethyl acetate (500 ml) and washed with saturated aqueous potassium carbonate. The ethyl acetate solution was separated, dried over sodium sulfate, and concentrated under vacuum to give an oil. This oil was Kugelrohr distilled under high vacuum to give tris(2-acetoxyethyl)methane (45.3 g, 95.9%) as an oil. Boiling point 220°C at 0.1 mmHg. [Table 4]

[0082] Step 1(d): Removal of the acetate group from the triacetate. Tris(2-acetoxyethyl)methane (45.3 g, 165 mM) in methanol (200 ml) and 880 ammonia (100 ml) was heated in an oil bath at 80° C. for 2 days. The reaction was treated with a further portion of 880 ammonia (50 ml) and heated in an oil bath at 80° C. for 24 hours. A further portion of 880 ammonia (50 ml) was added and the reaction heated at 80° C. for 24 hours. The reaction was then concentrated under vacuum and all solvent removed to give an oil. This was taken up in 880 ammonia (150 ml) and heated at 80° C. for 24 hours. The reaction was then concentrated under vacuum and all solvent removed to give an oil. Kugelrohr distillation gave acetamide, bp 170-180° C., 0.2 mm. The bulb containing the acetamide was cleaned and the distillation continued. Tris(2-hydroxyethyl)methane (22.53 g, 92%) was distilled at bp 220° C. and 0.2 mm. [Table 5]

[0083] Step 1(e): Conversion of the triol to the tris(methanesulfonate). To a stirred, ice-cold solution of tris(2-hydroxyethyl)methane (10 g, 0.0676 mol) in dichloromethane (50 ml) was added a solution of methanesulfonyl chloride (40 g, 0.349 mol) in dichloromethane (50 ml) slowly dropwise under nitrogen at such a rate that the temperature did not rise above 15° C. Then pyridine (21.4 g, 0.27 mol, 4 eq) dissolved in dichloromethane (50 ml) was added dropwise at such a rate that the temperature did not rise above 15° C. (exothermic reaction). The reaction was stirred at room temperature for 24 hours before being treated with 5N hydrochloric acid solution (80 ml) and the layers separated. The aqueous layer was extracted with more dichloromethane (50 ml) and the organic extracts were combined, dried over sodium sulfate, filtered and concentrated under vacuum to give tris[2-(methylsulfonyloxy)ethyl]methane contaminated with excess methanesulfonyl chloride. The theoretical yield was 25.8 g. [Table 6]

[0084] Step 1(f): Preparation of 1,1,1-tris(2-azidoethyl)methane. A stirred solution of tris[2-(methylsulfonyloxy)ethyl]methane [contaminated with excess methylsulfonyl chloride from step 1(e)] (25.8 g, 67 mmol, theoretical) in dry DMF (250 ml) was treated portionwise under nitrogen with sodium azide (30.7 g, 0.47 mol) over 15 minutes. An exotherm was observed and the reaction was cooled in an ice bath. After 30 minutes the reaction mixture was heated in a 50° C. oil bath for 24 hours. The reaction turned brown. The reaction was cooled, treated with dilute potassium carbonate solution (200 ml) and extracted three times with 40 / 60 petroleum ether / diethyl ether 10:1 (3×150 ml). The organic extract was washed with water (2×150 ml), dried over sodium sulfate and filtered. Ethanol (200 ml) was added to the petroleum / ether solution to keep the triazide in solution and the volume reduced by 200 ml under vacuum. Ethanol (200 ml) was added and reconcentrated under vacuum to remove the last traces of petroleum, leaving an ethanol solution of 200 ml. The ethanol solution of triazide was used directly in step 1(g).

[0085] Note: Do not remove all the solvent as the azide is potentially explosive and should always be kept in dilute solution.

[0086] Less than 0.2 ml of the solution was evaporated under vacuum to remove the ethanol and NMR was run on this small sample: [Table 7]

[0087] Step 1(g): Preparation of 1,1,1-tris(2-aminoethyl)methane. Tris(2-azidoethyl)methane (15.06 g, 0.0676 mol) in ethanol (200 ml) (assuming 100% yield from previous reaction) was treated with 10% palladium on charcoal (2 g, 50% water) and hydrogenated for 12 hours. Every 2 hours the reaction vessel was evacuated to remove the nitrogen evolved from the reaction and refilled with hydrogen. A sample was taken for NMR analysis which confirmed complete conversion of the triazide to the triamine.

[0088] CAUTION: Unreduced azide may explode during distillation. The reaction was filtered through a Celite pad to remove the catalyst and concentrated under vacuum to give tris(2-aminoethyl)methane as an oil, which was further purified by Kugelrohr distillation (bp 180-200 °C at 0.4 mm / Hg) to give a colorless oil (8.1 g, 82.7% overall yield). [Table 8]

[0089] Example 2: Preparation of 3-chloro-3-methyl-2-nitrosobutane. A mixture of 2-methylbut-2-ene (147 ml, 1.4 mol) and isoamyl nitrite (156 ml, 1.16 mol) was cooled to -30°C in a bath of dry ice and methanol, stirred vigorously with an overhead air stirrer, and treated dropwise with concentrated hydrochloric acid (140 ml, 1.68 mol) at such a rate that the temperature was maintained below -20°C. This takes approximately 1 hour, as there is a significant exotherm, and care must be taken to prevent overheating. Ethanol (100 ml) was added to reduce the viscosity of the slurry formed at the end of the addition, and the reaction was stirred at -20 to -10°C for a further 2 hours to drive the reaction to completion. The precipitate was collected by filtration under vacuum, washed with 4 x 30 ml of cold (-20°C) ethanol and 100 ml of ice-cold water, and dried under vacuum to give 3-chloro-3-methyl-2-nitrosobutane as a white solid. The ethanol filtrate and washings were combined, diluted with water (200 ml), and cooled and allowed to stand at -10°C for 1 h, which caused further crystallization of 3-chloro-3-methyl-2-nitrosobutane. The precipitate was collected by filtration, washed with a minimum of water, and dried under vacuum to give a total yield of 3-chloro-3-methyl-2-nitrosobutane (115 g, 0.85 mol, 73%) with a purity of >98% by NMR. [Table 9]

[0090] Example 3: Synthesis of bis[N-(1,1-dimethyl-2-N-hydroxyiminepropyl) 2-aminoethyl]-(2-aminoethyl)methane (chelating agent 1). To a solution of tris(2-aminoethyl)methane (Example 1; 4.047 g, 27.9 mmol) in dry ethanol (30 ml) was added anhydrous potassium carbonate (7.7 g, 55.8 mmol, 2 equiv.) at room temperature under nitrogen atmosphere with vigorous stirring. A solution of 3-chloro-3-methyl-2-nitrosobutane (Example 2; 7.56 g, 55.8 mol, 2 equiv.) was dissolved in dry ethanol (100 ml) and 75 ml of this solution was slowly added dropwise to the reaction mixture. After the reaction, it was analyzed by TLC on silica [dichloromethane, methanol, concentrated ammonia (0.88 sg); plates were run with 100 / 30 / 5 and TLC plates were developed by spraying with ninhydrin and heating]. Mono-, di- and tri-alkylated products were seen and RF' increased in that order. Analytical HPLC was performed using a PRP reversed phase column with a gradient of 7.5-75% acetonitrile in 3% aqueous ammonia. The reaction was concentrated under vacuum to remove the ethanol and resuspended in water (110 ml). The aqueous slurry was extracted with ether (100 ml) to remove the trialkylated compound and some of the lipophilic impurities, leaving the mono and desired dialkylated products in the aqueous layer. To ensure good chromatography, the aqueous solution was buffered with ammonium acetate (2 equiv., 4.3 g, 55.8 mmol). The aqueous solution was stored at 4° C. overnight before being purified by automated preparative HPLC. Yield (2.2 g, 6.4 mmol, 23%). Mass spectrometry; positive ion 10V cone voltage. Found: 344; calculated M+H=344. [Table 10]

[0091] HPLC conditions: flow rate 8 ml / min, using a 25 mm PRP column [A=3% ammonia solution (sp.gr=0.88) / water; B=acetonitrile]. [Table 11]

[0092] Example 4: Synthesis of chelating agent 1 - tetrafluorothiophenyl ester of glutaric acid (chelating agent 1A). (Step 4a) Synthesis of [chelating agent 1]-glutaric acid intermediate. [ka]

[0093] Chelating agent 1 (100 mg, 0.29 mmol) was dissolved in DMF (10 ml) and glutaric anhydride (33 mg, 0.29 mmol) was added portionwise with stirring. The reaction was stirred for 23 h to achieve complete conversion to the desired product. The pure acid was obtained in good yield after RP-HPLC.

[0094] (Step 4b) Synthesis of chelating agent 1A. [ka]

[0095] To [chelator 1]-glutaric acid (from step 4a; 300 mg, 0.66 mmol) in DMF (2 ml) was added HATU (249 mg, 0.66 mmol) and NMM (132 μL, 1.32 mmol). The mixture was stirred for 5 min, then tetrafluorothiophenol (0.66 mmol, 119 mg) was added. The solution was stirred for 10 min, then the reaction mixture was diluted with 20% acetonitrile / water (8 ml) and the product was purified by RP-HPLC to give 110 mg of the desired product after lyophilization.

[0096] Example 5: Disulfide [Cys 2-6 ]Thioethercyclo[CH 2 CO-Lys(chelating agent 1-glutaryl)-Cys 2 -Arg-Gly-Asp-Cys 6 -Phe-Cys]-NH 2 Synthesis of. [ka]

[0097] (Step 5a) ClCH 2 CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-NH 2 Synthesis of [ka]

[0098] Peptides were synthesized on an ABI 433A automated peptide synthesizer on a 0.25 mmol scale starting with Rink Amide AM resin using a 1 mmol amino acid cartridge. Amino acids were preactivated with HBTU prior to coupling. The N-terminal amine group was chloroacetylated with chloroacetic anhydride in DMF for 30 min. Simultaneous removal of the peptide and side chain protecting groups (except tBu) from the resin was achieved using TIS (5%), H 2 The reaction was carried out in TFA containing HO (5%) and phenol (2.5%) for 2 h. After workup, 295 mg of crude peptide was obtained (analytical HPLC: gradient, 5-50% B in 10 min, where A=HO). 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 6.42 min. The product was further characterized using mass spectrometry: expected M+H 1118.5, observed 1118.6).

[0099] (Step 5b) Thioether cyclo[CH 2 CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-NH 2 Synthesis of. [ka]

[0100] 295 mg ClCH 2 CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-NH 2 was dissolved in water / acetonitrile. The mixture was adjusted to pH 8 with ammonia solution and stirred for 16 h. After workup, 217 mg of crude peptide was obtained (analytical HPLC: gradient, 5-50% B in 10 min, where A=H 2 O / 0.1% TFA and B=CH 3CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 6.18 min. The product was further characterized using mass spectrometry: expected M+H 1882.5, observed 1882.6).

[0101] (Step 5c) Disulfide [Cys 2-6 ]Thioethercyclo[CH 2 CO-Lys-Cys 2 -Arg-Gly-Asp-Cys 6 -Phe-Cys]-NH 2 Synthesis of. [ka]

[0102] 217mg of thioether cyclo[CH 2 CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-NH 2 The peptide was treated with a solution of anisole (500 μL), DMSO (2 ml) and TFA (100 ml) for 60 min, after which the TFA was removed under vacuum and diethyl ether was added to precipitate the peptide. Purification of the crude material (202 mg) by preparative HPLC (Phenomenex Luna 10 μC18(2) 250 × 50 m column) was performed using 0–30% B (where A=H 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA) at a flow rate of 50 ml / min for 60 min. After lyophilization, 112 mg of pure material was obtained (analytical HPLC: gradient, 5-50% B in 10 min, where A=H 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 5.50 min. The product was further characterized using mass spectrometry: expected M+H 968, observed 971).

[0103] (Step 5d) Disulfide [Cys 2-6 ]Thioethercyclo[CH 2 CO-Lys(chelating agent 1-glutaryl)-Cys 2 -Arg-Gly-Asp-Cys 6 -Phe-Cys]-NH 2 Synthesis of. [ka]

[0104] 9.7 mg of disulfide [Cys 2-6 ]Thioethercyclo[CH 2 CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-NH 2 To this end, 9.1 mg of chelating agent 1A (Example 5) and 6 μL of NMM were dissolved in DMF (0.5 ml). The mixture was stirred for 3 h. Purification of the reaction mixture by preparative HPLC (Phenomenex Luna 5μC18(2) 250×21.20 mm column) was performed using 0-30% B (where A=H 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA) at a flow rate of 10 ml / min for 40 min. After lyophilization, 5.7 mg of pure material was obtained (Analytical HPLC: gradient, 0-30% B in 10 min, where A=H 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 7.32 min. The product was further characterized using mass spectrometry: expected M+H 1407.7, observed 1407.6.

[0105] Example 6: Disulfide [Cys 2-6 ]Thioethercyclo[CH 2 CO-Lys(chelating agent 1-glutaryl)-Cys 2 -Arg-Gly-Asp-Cys 6 -Phe-Cys]-(PEG) 3 -NH2 Synthesis of (malaciclactide). (Step 6a) Synthesis of 17-(Fmoc-amino)-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid. [ka]

[0106] The building blocks are attached to the solid phase using Fmoc chemistry.

[0107] 1,11-Diazido-3,6,9-trioxaundecane. A solution of dry tetraethylene glycol (19.4 g, 0.100 mol) and methanesulfonyl chloride (25.2 g, 0.220 mol) in dry THF (100 ml) was kept under argon and cooled to 0° C. in an ice / water bath. To this flask was added a solution of triethylamine (22.6 g, 0.220 mol) in dry THF (25 ml) dropwise over 45 min. After 1 h, the cooling bath was removed and stirring was continued for 4 h. Water (60 ml) was added. To the mixture were added sodium bicarbonate (6 g, to pH 8) and sodium azide (14.3 g, 0.220 mmol) in that order. THF was removed by distillation and the aqueous solution was refluxed for 24 h (two layers formed). The mixture was cooled and ether (100 ml) was added. The aqueous phase was saturated with sodium chloride. The phases were separated and the aqueous phase was extracted with ether (4×50 ml). The combined organic phase was washed with brine (2×50 ml) and dried (MgSO 4 Filtration and concentration gave 22.1 g (91%) of a yellow oil which was used in the next step without further purification.

[0108] 11-Azido-3,6,9-trioxaundecanamine. To a vigorously mechanically stirred suspension of 1,11-diazide-3,6,9-trioxaundecane (20.8 g, 0.085 mol) in 5% hydrochloric acid (200 ml) was added a solution of triphenylphosphine (19.9 g, 0.073 mol) in ether (150 ml) over 3 h at room temperature. The reaction mixture was stirred for a further 24 h. The phases were separated and the aqueous phase was extracted with dichloromethane (3 x 40 ml). The aqueous phase was cooled in an ice / water bath and the pH was adjusted to approximately 12 by the addition of KOH. The product was extracted into dichloromethane (5 x 50 ml). The combined organic phase was dried (MgSO 4 Filtration and evaporation gave 14.0 g (88%) of a yellow oil. Analysis by MALDI-TOF mass spectrometry (matrix: α-cyano-4-hydroxycinnamic acid) gave the expected M+H peak at 219. 1 H(500MHz) and 13 Further characterization using C (125 MHz) NMR spectroscopy confirmed the structure.

[0109] 17-Azido-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid. To a solution of 11-azido-3,6,9-trioxaundecanamine (10.9 g, 50.0 mmol) in dichloromethane (100 ml) was added diglycolic anhydride (6.38 g, 55.0 mmol). The reaction mixture was stirred overnight. HPLC analysis (column Vydac 218TP54; solvent: A=water / 0.1% TFA and B=acetonitrile / 0.1% TFA; gradient, 4-16% B in 20 min; flow rate 1.0 ml / min; UV detection at 214 nm and 284 nm) showed complete conversion of starting material to product with a retention time of 18.3 min. The solution was concentrated to give a yellow syrup in quantitative yield. The product was analyzed by LC-MS (ES ionization) showing [MH] at 335 as expected. + was obtained. 1 H(500MHz) and 13 C (125 MHz) NMR spectroscopy was consistent with structure (I). The product was used in the next step without further purification.

[0110] 17-Amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid. A solution of 17-azido-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid (8.36 g, 25.0 mmol) in water (100 mL) was 2 (g)-Pd / C (10%) was used for reduction. The reaction was run until complete conversion of starting material was confirmed by LC-MS analysis (column Vydac 218TP54; solvent: A=water / 0.1% TFA and B=acetonitrile / 0.1% TFA; gradient, 4-16% B in 20 min; flow rate 1.0 ml / min; UV detection at 214 nm and 284 nm, ES ionization gave M+H at 335 for starting material and 309 for product). The solution was filtered and used directly in the next step.

[0111] 17-(Fmoc-amino)-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid. To an aqueous solution of 17-amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid from above (corresponding to 25.0 mmol of amino acid) was added sodium bicarbonate (5.04 g, 60.0 mmol) and dioxane (40 ml). A solution of Fmoc-chloride (7.11 g, 0.275 mol) in dioxane (40 ml) was added dropwise. The reaction mixture was stirred overnight. Dioxane was removed by evaporation (rotavapor) and the aqueous phase was extracted with ethyl acetate. The aqueous phase was acidified with hydrochloric acid and the precipitate was extracted into chloroform. The organic phase was dried (MgSO 4 ), filtered and concentrated to give 11.3 g (85%) of a yellow syrup. The structure was confirmed by LC-MS analysis (column Vydac 218TP54; solvents: A=water / 0.1% TFA and B=acetonitrile / 0.1% TFA; gradient, 40-60% B in 20 min; flow rate 1.0 ml / min; UV detection at 214 nm and 254 nm, ES ionization gave M+H at 531 as expected for the product peak at 5, 8 min). Analysis showed very low content of by-products, so this material was used without further purification.

[0112] (Step 6b)ClCH 2 CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG) 3 -NH 2 Synthesis of. [ka]

[0113] The PEG unit was manually coupled to Rink Amide AM resin via HATU activation starting at a 0.25 mmol scale. The remaining peptide was assembled on an ABI 433A automated peptide synthesizer using 1 mmol amino acid cartridges. Amino acids were pre-activated with HBTU prior to coupling. The N-terminal amine group was chloroacetylated using a solution of chloroacetic anhydride in DMF for 30 min.

[0114] The simultaneous removal of peptides and side chain protecting groups (except tBu) from the resin was performed using TIS (5%), H 2 The reaction was carried out in TFA containing HO (5%) and phenol (2.5%) for 2 h. After workup, 322 mg of crude peptide was obtained (analytical HPLC: gradient, 5-50% B in 10 min, where A=HO). 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 6.37 min. The product was further characterized using mass spectrometry: expected M+H 1409, observed 1415.

[0115] (Step 6c) Thioether cyclo[CH 2 CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG) 3 -NH 2 Synthesis of (NC100717). See Indrevoll et al. [Bioorg. Med. Chem. Lett., 16, 6190-6193 (2006)]. [ka]

[0116] 322 mg of ClCH 2CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG) 3 -NH 2 was dissolved in water / acetonitrile. The mixture was adjusted to pH 8 with ammonia solution and stirred for 16 h.

[0117] After workup, the crude peptide was obtained (Analytical HPLC: gradient, 5-50% B in 10 min, where A=H 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 6.22 min. The product was further characterized using mass spectrometry: expected M+H 1373, observed 1378.

[0118] (Step 6d) Disulfide [Cys 2-6 ]Thioethercyclo[CH 2 CO-Lys-Cys 2 -Arg-Gly-Asp-Cys 6 -Phe-Cys]-(PEG) 3 -NH 2 Synthesis of. [ka]

[0119] Thioethercyclo[CH 2 CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG) 3 -NH 2 The peptide was treated with a solution of anisole (200 μL), DMSO (2 ml) and TFA (100 ml) for 60 min, after which the TFA was removed under vacuum and diethyl ether was added to precipitate the peptide. Purification of 70 mg of the crude material by preparative HPLC (Phenomenex Luna 5μC18(2) 250×21.20 mm column) was carried out using 0–30% B (where A=H 2 O / 0.1% TFA and B=CH3 CN / 0.1% TFA) at a flow rate of 10 ml / min for 40 min. After lyophilization, 46 mg of pure material was obtained (analytical HPLC: gradient, 0-30% B in 10 min, where A=H 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 6.80 min. The product was further characterized using mass spectrometry: expected M+H 1258.5, observed 1258.8).

[0120] (Step 6e) Disulfide [Cys 2-6 ]Thioethercyclo[CH 2 CO-Lys(chelating agent 1-glutaryl)-Cys 2 -Arg-Gly-Asp-Cys 6 -Phe-Cys]-(PEG) 3 -NH 2 Synthesis of. [ka]

[0121] 13mg [Cys 2-6 ]Cyclo[CH 2 CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-(PEG) 3 -NH 2 9.6 mg of chelator 1A and 8 μL of NMM were dissolved in DMF (0.5 ml). The mixture was stirred for 2 h 30 min. Purification of the reaction mixture by preparative HPLC (Phenomenex Luna 5μC18(2) 250×21.20 mm column) was performed using 0–30% B (where A=H 2 O / 0.1% TFA and B=CH 3 CN / 0.1% TFA) at a flow rate of 10 ml / min for 40 min. After lyophilization, 14.2 mg of pure material was obtained (analytical HPLC: gradient, 0-30% B in 10 min, where A=H 2 O / 0.1% TFA and B=CH 3CN / 0.1% TFA; column, Phenomenex Luna 3μC18(2) 50×4.6 mm; flow rate, 2 ml / min; detection, UV 214 nm; product retention time, 7.87 min. The product was further characterized using mass spectrometry: expected M+H 1697.8, observed 1697.9.

[0122] Example 7: Preparation of Malaciclactide Lyophilized Non-Radioactive Kit. A lyophilized kit was prepared having the following formulation: [Table 12]

[0123] Malaciclatide and excipients are dissolved in water for injection under a nitrogen atmosphere. Aliquots are dispensed into glass vials after sterile filtration and synthetic rubber stoppers are added. The solution is then frozen at -42°C and lyophilized under vacuum to remove the water. The lyophilized material is then sealed under a nitrogen atmosphere and sealed with an aluminum overseal. Vials can be stored for long periods at 2-8°C before use.

[0124] Example 8: 99m Preparation of Tc-malaciclactide. The kit of Example 7 is reconstituted under sterile conditions with up to 3.1 GBq / 6 ml of generator eluate from a commercial technetium generator and left at room temperature for 20 minutes. Quality control is performed by both visual assessment and measurement of radiochemical purity (RCP) by thin layer chromatography using silica-coated paper and a mobile phase containing 50:50 methanol and 1 molar ammonium acetate. The reconstituted solution is colorless and free of visible particles with an RCP of greater than 85%. Sufficient material to provide a patient dose is drawn into a syringe under sterile conditions.

[0125] Example 9: Uptake in animal models of endometriosis. A rat surgical model of endometriosis was used along with an immunodeficient mouse model and a syngeneic mouse model. All three animal models of endometriosis were as described by Story, L. ILAR J., 45(2) 132-138 (2004).

[0126] 99m Tc-malaciclactide uptake 99m The uptake of Tc-NC100677 was compared with that of Tc-NC100677. NC100667 was a novel cytoplasmic ... 35 , 365-375 (2008)]. NC100667 is a negative control peptide in which the arginine residue in the RGD binding motif of malaciclatide (NC100692) is replaced with an alanine residue. Thus, only one amino acid residue was changed; all other aspects of the drug were otherwise the same. The results are shown in Figures 1-4.

[0127] 99m Uptake and retention of Tc-malaciclactide was demonstrated in all experimental animal models of endometriosis.

Claims

1. 1. A method of imaging areas of endometriosis in a subject, comprising: In vivo in said subject comprising 99mTc-malaciclatide radiopharmaceutical. ９９ｍ ex utero imaging of radioactive emissions from a Tc-malaciclatide radiopharmaceutical, wherein the site is: (i) Abdominal endometriosis; (ii) peritoneal endometriosis; (iii) thoracic endometriosis; An imaging method comprising one or more of:

2. The imaging method of claim 1 , wherein the site comprises a superficial endometriotic lesion.

3. The imaging method of claim 1 , wherein the site comprises abdominal or peritoneal endometriosis.

4. The imaging method of claim 1 , wherein superficial endometriotic lesions are imaged in combination with peritoneal endometriosis.

5. 2. The imaging method of claim 1, wherein all three of (i) through (iii) are imaged via whole-body imaging of the subject.

6. 10. The imaging method of claim 1, wherein the radioactive emissions are detected and processed using a gamma camera; a gamma detector; image processing software, or a combination thereof.

7. 7. The imaging method of claim 6, wherein the gamma camera is a high-resolution gamma camera having a spatial resolution of less than 1 millimeter.

8. The imaging method of claim 7 , wherein the high resolution gamma camera includes a CZT detector.

9. The aforementioned ９９ｍ The Tc-malaciclatide radiopharmaceutical is prepared by converting malaciclatide to HCl in the presence of a reducing agent. ９９ｍ 2. The imaging method of claim 1, wherein the compound is prepared by reacting with Tc-pertechnetate.

10. The aforementioned ９９ｍ Tc-malaciclatide radiopharmaceutical is available in non-radioactive kits. ９９ｍ 10Tc-pertechnetate solution, wherein the non-radioactive kit is prepared by reconstituting the kit with a solution of 10Tc-pertechnetate. a) Malaciclatide; b) para-aminobenzoic acid or a salt thereof with a biocompatible cation; c) a stannous reducing agent; d) methylenediphosphonic acid or its salt with a biocompatible cation The imaging method of claim 1 , comprising:

11. A method for determining treatment for endometriosis in a subject, comprising the imaging method described in any one of claims 1 to 10.

12. A method for monitoring the treatment of endometriosis in a subject, comprising the imaging method described in any one of claims 1 to 10.

13. One or more of the following: (i) an imaging method according to any one of claims 1 to 10; (ii) a method for diagnosing endometriosis, comprising the imaging method of (i); (iii) a method for determining treatment of endometriosis in a subject, the method comprising the imaging method of (i); (iv) a method for monitoring the treatment of endometriosis in a subject, the method comprising the imaging method of (i); A kit for the preparation of 99m Tc-malaciclatide for use in A non-radioactive malaciclatide kit, which is a non-radioactive kit as defined in claim 10.

14. One or more of the following: (i) an imaging method according to any one of claims 1 to 10; (ii) a method for determining treatment of endometriosis in a subject, the method comprising the imaging method of (i); (iii) a method for monitoring treatment of endometriosis in a subject, the method comprising the imaging method of (i); The use of a gamma camera, gamma detector and / or image processing software in 15. A 99m Tc-malaciclatide radiopharmaceutical for diagnosing endometriosis, comprising 99m Tc-malaciclatide and used for diagnosing endometriosis, the diagnosis being carried out including the imaging method according to any one of claims 1 to 10.

Images