Molecular imaging complex for positron emission tomography

Abstract

The present invention discloses complex for PET imaging in patients with Cushing's syndrome dependent on secretion of a hormone called ACTH from pituitary. This will help to distinguish Cushing's disease due to pituitary tumor (adenoma) or due to tumor of origin other than pituitary (ectopic Cushing's syndrome). It will also be helpful to delineate ACTH producing pituitary adenoma (corticotropinoma). More particularly the work disclosed an imaging complex comprising radiolabelled analogue of desmopressin for PET imaging to localize pituitary adenoma.

Claims

1. A molecular imaging complex comprising desmopressin analogue and a bifunctional chelator for tomography imaging wherein a. desmopressin analogue has D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly (disulfide cyclized Cys2-Cys7) sequence; b. desmopressin analogue and bifunctional chelator are in 1:1 molar ratio; and c. bifunctional chelator is conjugated with desmopressin analogue through covalent bond.

2. The molecular imaging complex as claimed in claim 1, wherein bifunctional chelator is selected from group comprising DOTA, DATA, NOTA, DOTAGA, NODAGA, DTPA, HBED-CC.

3. The molecular imaging complex as claimed in claim 1, is further conjugated with a radioisotope by heating the said complex with a radioisotope at 90-100 C. for 5-30 min at pH 3.5-4.5, followed by purification with solid phase extraction chromatography.

4. The molecular imaging complex as claimed in claim 3, wherein radioisotope is selected from the group consisting of Gallium-68, Indium-111, Yttrium-90, Lutitium-177, Zirconium-89, Copper-64.

5. The molecular imaging complex as claimed in above claims is used for differential diagnosis and localization of ACTH dependent Cushing's syndrome.

Description

BRIEF DESCRIPTION OF FIGURES

[0009] The present invention will become more understandable from the description given herein and the accompanying drawings below. These are given by way of illustration only and therefore not limited to present invention and wherein:

[0010] FIG. 1: Intensity v/s distance plot of .sup.68Ga-mDesmo obtained with radio-TLC scanner using whatman paper 3 as a stationary phase and sodium citrate as mobile phase. Single peak of .sup.68Ga-mDesmo with R.sub.f value 0.1, shows >99% radiochemical purity

[0011] FIG. 2: MALDI-TOF of DOTA-D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Thr(ol) (disulfide cyclized Cys2-Cys7) with molecular weight 1648.89.

[0012] FIG. 3: HPLC of DOTA-D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Thr(ol) (disulfide cyclized Cys2-Cys7)

[0013] FIG. 4: a) MIP of Ga-68 mDesmo (radiotracer) PET showing no uptake in any organ throughout the body. Visualization of kidney and bladder demonstrating renal route of excretion of radiotracer, b) CT brain for anatomical details c) PET/CT fused image of brain and pituitary showing no radiotracer uptake.

[0014] FIG. 5: A patient with macroadenoma on a) CT showing macroadenoma; b) PET and c) PET/CT fused images showing intense uptake (SUVmax 3.7) of radiotracer confirming specificity of radiotracer for pituitary adenoma.

BEST MODE(S) FOR CARRYING OUT THE INVENTION

[0015] The following presents a simplified description of the invention in order to provide a basic understanding of some aspects of the invention. This description is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form.

[0016] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.

[0017] Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.

[0018] The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the invention as defined by the appended claims and their equivalents.

[0019] It is to be understood that the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise.

[0020] By the term substantially it is meant that the recited characteristic, parameter, or value need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement error, measurement accuracy limitations and other factors known to those of skill in the art, may occur in amounts that do not preclude the effect the characteristic was intended to provide.

[0021] It should be emphasized that the term comprises/comprising when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0022] The term radiolabeling disclosed herein refers to the binding of radionuclide with molecule of interest such as peptide.

[0023] The term DOTA/NOTA/DOTAGA/NODAGA disclosed herein refers to the organic compound used as a bifunctional chelating agent for positron emitting isotopes for PET imaging.

[0024] It should be emphasized that the term Desmopressin disclosed herein refers to the 1-(3-mercaptopropanoic acid or Mpr)-Tyr-Phe-Gln-Asn-Cys-Pro-D Arg-Gly-NH2.

[0025] It should be emphasized that the term mDesmo disclosed herein refers to the complex of Desmopressin with bifunctional chelator DOTA.

[0026] It should be emphasized that the term .sup.68Ga-mDesmo disclosed herein refers to the radiolabelling of mDesmo with Ga-68 radioisotope.

[0027] It should be emphasized that the term DOTA when used in this specification is taken to specify the 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0028] It should be emphasized that the term DATA when used in this specification is taken to specify the ((6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate).

[0029] It should be emphasized that the term NOTA when used in this specification is taken to specify the 1,4,7-triazacyclononane-N,N,N-triacetic acid

[0030] It should be emphasized that the term DOTAGA when used in this specification is taken to specify the 2-[1,4,7,10-Tetraazacyclododecane-4,7,10-tris(t-butyl acetate)]-pentanedioic acid-it-butyl ester.

[0031] It should be emphasized that the term NODAGA when used in this specification is taken to specify the 2-[1,4,7-Triazacyclononan-1-yl-4,7-bis(tBu-ester)]-1,5-pentanedioic acid

[0032] It should be emphasized that the term PET (Positron Emitting Tomography) disclosed herein refers to the nuclear medicine tomographic imaging technique using positrons.

[0033] It should be emphasized that the term MRI disclosed herein refers to the magnetic resonance imaging uses a strong magnetic field and radio waves to create detailed images of the organs and tissues within the body.

[0034] It should be emphasized that the term radiological imaging disclosed herein refers to the-imaging techniques such as X-ray radiography, ultrasound, computed tomography (CT), nuclear medicine including positron emission tomography (PET), and magnetic resonance imaging (MRI) are used to diagnose or treat diseases

[0035] It should be emphasized that the term corticotropinoma disclosed herein refers to the pituitary adenoma made up predominantly of corticotrophs.

[0036] It should be emphasized that the term bifunctional chelator disclosed herein refers to the molecule that has ability to bind with biomolecule and has metal chelation property.

[0037] It should be emphasized that the term differential diagnosis disclosed herein refers to distinguish between Cushing's disease and ectopic Cushing's syndrome (ECS).

[0038] It should be emphasized that the term Seq disclosed herein refers to sequence of amino acids.

Embodiments

[0039] Desmopressin is a 9 amino acid long peptide bind to V2, V3 receptor. To develop a target specific ligand, it is of utmost importance to preserve the binding domain. The di-sulfide bridge between 2 cystein residue plays an important role in binding and stability of Desmopressin to the receptor.

[0040] Desmopressin has Seq-1(Mpr-Tyr-Phe-Gln-Asn-Cys(1)-Pro-D-Arg-Gly-NH2) with molecular weight 1069.

[0041] In an embodiment the Desmopressin analogue is prepared by adding/removing and/or modifying an amino acid at the N or/and C terminal of desmopressin.

[0042] In an embodiment the Desmopressin analogue comprising replacement of Mpr of Seq-1 with Cys, with Seq-2 (Cys-Tyr-Phe-Gln-Asn-Cys(1)-Pro-D-Arg-Gly-NH.sub.2).

[0043] In an embodiment the Desmopressin analogue (Seq-3) involves the addition D-Phe at C terminal of Seq-2, with Seq-3 (D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys(1)-Pro-D-Arg-Gly-NH2).

[0044] In an embodiment the Desmopressin analogue (Seq-4) involves the replacement of Gly-NH.sub.2 with Thr(ol) at N terminal of Seq-3, with Seq-3 (D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys(1)-Pro-D-Arg-Gly-NH.sub.2).

[0045] In a preferred embodiment the Desmopressin analogue (mDesmo) is a 10 amino acid peptide with the sequence: (Seq-4: D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Thr(ol) (disulfide cyclized Cys2-Cys7).

[0046] Conjugation of desmopressin analogue with bifunctional chelator (mDesmo): The desmopressin analogue (Seq-4) is of ten amino acid is conjugated with a bifunctional chelator.

[0047] In an embodiment the bifunctional chelator is conjugated to D-Phe.

[0048] In an embodiment the the bifunctional chelator is selected from the group comprising of DOTA, DATA, DOTAGA, NOTA, NOTAGA, NODAGA, Cyclic DTPA.

[0049] In an embodiment the molecular weight is mDesmo is 1648.89, when bifunctional chelator is DOTA.

[0050] In an embodiment FIG. 2 Illustrating MALDI-TOF of DOTA-D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Thr(ol) (disulfide cyclized Cys2-Cys7) showing peak at 550.7 Da, 825.5 Da and a small peak at 523.4

[0051] In an embodiment FIG. 3 Illustrating HPLC of DOTA-D-Phe-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Thr(ol) (disulfide cyclized Cys2-Cys7) With retention time 12.77 minute.

[0052] Radiolabeling of mDesmo with .sup.68Ga: For radiolabelling of mDesmo), .sup.68Ga is eluted from .sup.68Ge-.sup.68Ga generator (iQS .sup.68Ga Fluidic Labelling Module, itG, Germany). For standardization of radiolabeling parameters, various combinations of different reaction parameters such as pH (1-14), amount of peptide (1-100 g), concentration and volume of sodium acetate buffer corresponding to .sup.68Ga eluted with 4 mL of 0.05M HCl, incubation time (2-30 min) and temperature (25-100 C.) have been evaluated.

[0053] Purification: The residual free radionuclide (.sup.68Ga) in the reaction mixture, if present, is subjected to removal as it gets accumulated in non-targeted organs thereby, altering the biodistribution, which ultimately leads to unnecessary radiation burden to the patient.

[0054] In an embodiment after radiolabeling, the reaction mixture is subjected to the purification using solid phase extraction (SPE) method with Sep-Pak C18 cartridges.

[0055] In an embodiment the C18 cartridge is first conditioned using 70% ethanol (5 mL) followed by washing with 10 mL water (HPLC grade) at the flow rate of 1-2 mL/min. The radiolabeled mixture is allowed to pass through cartridge enabling the hydrophobic interaction between the peptide and carbon chain of C18 cartridge. The trapped radiolabeled peptide is finally eluted with 1 mL of 50% ethanol with the flow rate of 1 m/min.

[0056] Radiochemical Purity: Radio thin-layer chromatography (radio-TLC) is performed to assess the radiochemical purity (RCP) of the in-house synthesized radiopharmaceutical (.sup.68Ga-mDesmo). Various combinations of mobile phase solvents and stationary phases are assessed for their potential to demarcate the desired radiolabelled product (.sup.68Ga-mDesmo) from free radionuclide (.sup.68Ga) in the final reaction mixture. The R.sub.f values of .sup.68Ga-mDesmo, and .sup.68Ga are determined for each solvent and stationary phase using Radio-TLC scanner.

[0057] Stability: Shelf-life of .sup.68Ga-mDesmo is assessed at room temperature up to 6 hours. Radio-chromatograms are obtained at various time points such as 30 min, 60 min, 120 min, 180 min, and 240 min using whatman paper 3 and sodium citrate as a stationary phase and mobile phase, respectively. In addition to that the stability of in house synthesized .sup.68Ga-mDesmo is assessed in human serum at 37 C. for 4 hr.

[0058] In an embodiment the radiolabeling yield of >99% has been obtained when 5-25 mCi of .sup.68Ga incubated with 1-100 g of mDesmo at 70-100 C. for 2-30 min. The reaction pH is maintained at 1-14 using sodium acetate buffer and after incubation the crude reaction mixture is purified using C18 cartridge.

[0059] In a preferred embodiment the radiolabeling yield of >99% has been obtained when 25 mCi of .sup.68Ga incubated with 20 g of DOTA-DDAVP at 95 C. for 10 min. The reaction pH is maintained at 3.5-4.5 using 0.025 M sodium acetate buffer and after incubation the crude reaction mixture is purified using C18 cartridge. The product showed shelf life of 4 hr at room temperature, and was found stable in human serum stability up to 4 hr at 37 C.

[0060] In a embodiment the radioisotope is selected from the group consisting of Gallium-68, Indium-111, Yttrium-90, Lutitium-177, Zirconium-89, Copper-64.

[0061] Clinical Studies: The patients suspected with Cushing's syndrome are enrolled from the endocrine clinic of PGIMER, Chandigarh. The detailed patient history is taken, and physical examination is done. The complete hemogram and biochemistry are taken as baseline investigations. Hormonal evaluation includes serum cortisol obtained at 0800 h and 1100 h. Dexamethasone suppression test (LDDST): A dose of 0.5 mg dexamethasone is to be administered orally strictly every 6-hour interval for 48 h. Blood samples are collected for serum cortisol measurement at 0800 h on Day 3 following the first dose of dexamethasone. High dose dexamethasone suppression test (HDDST): Dexamethasone at a dose of 2 mg is to be administered orally strictly every 6 hours for 48 hours. Blood sample is collected for serum cortisol measurement at 0800 hr on Day 3 following the first dose of dexamethasone.

[0062] ACTH Induction Studies: Injection of 10 g conjugated mDesmo intravenously is followed by estimation of ACTH and cortisol level after (15, 30, 45 and 60 min minutes to look for in-vivo efficiency of conjugated molecule to increase ACTH and cortisol levels.

[0063] PET/CT Imaging: An activity of 57 Ci/kg body weight of .sup.68Ga-mDesmo (2-4 mCi) is administered intravenously in all patients. For three patients dynamic PET/CT imaging of brain is performed for 60 minutes with a dedicated PET/CT scanner (Discovery MIDR, GE Healthcare, USA) to observe maximum uptake time in the pituitary (sella) region. For rest of the patients, PET imaging will be performed for 10 minutes at optimum uptake time derived from dynamic studies to localize corticotropinoma. For all patients CT is acquired first followed by PET acquisition. The CT acquisition parameters used are tube voltage of 140 kVp, tube current (100-350 mA), 0.625 mm helical thickness, 0.8 s rotation time, and 0.531:1 pitch.

[0064] PET/CT processing protocol: The CT images were reconstructed in a matrix of 512512 with a slice thickness of 0.625 mm. Data obtained from CT acquisition is used for attenuation correction of PET emission data and for fusion of attenuation corrected PET images with corresponding CT images. PET images were reconstructed in matrix of 384384 using iterative reconstruction algorithm OSEM (32 subsets and 8 iterations). The reconstructed attenuation corrected PET images, CT images and fused images of matching pairs of PET and CT images are available for review in axial, coronal and sagittal planes, as well as in maximum intensity projections, three dimentional cine mode. A region of intrest (ROI) is carefully drawn around the site of the lesions. The slice with maximal uptake in the ROI is choosen for quantitative measurement of activity of the lesion (SUV). The SUV is calculated according to the formula described below.

SUV=Mean ROI activity(MBq/g)/Injected dose(MBq)/Body weight(g)

where, MBq=mega-Becquerel, and g=grams

[0065] As illustrated in FIG. 4. a) MIP of Ga-68 mDesmo (radiotracer) PET showing no uptake in any organ throughout the body. Visualization of kidney and bladder demonstrating renal route of excretion of radiotracer, b) CT brain for anatomical details c) PET/CT fused image of brain and pituitary showing no radiotracer uptake.

[0066] As illustrated in FIG. 5. A patient with macroadenoma on a) CT showing macroadenoma; b) PET and c) PET/CT fused images showing intense uptake (SUVmax 3.7) of radiotracer confirming specificity of radiotracer for pituitary adenoma.

[0067] The processes above are described as a sequence of steps, this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, the order of the steps may be re-arranged, or some steps may be performed simultaneously.

[0068] Although embodiments have been described with reference to specific example embodiments, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader spirit and scope of the system and method described herein. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

[0069] Many alterations and modifications of the present invention will no doubt become apparent to a person of ordinary skill in the art after having read the foregoing description. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. It is to be understood that the description above contains many specifications; these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the personally preferred embodiments of this invention.

INDUSTRIAL APPLICABILITY

[0070] The invention particularly relates to differential diagnosis of Cushing's syndrome i. e. to distinguish between Cushing's disease and ectopic Cushing's syndrome (ECS). Cushing's syndrome is a clinical condition characterized by excessive circulating cortisol and by various clinical features such as gain of weight, menstrual, disturbances, hirsutism, psychiatric dysfunction, proximal muscle weakness, osteoporosis, fractures, loss of scalp hair, plethora, rounding of face, hypertension, easy bruising, wide purple striae, (>1 cm) hyper pigmentation, and diabetes. Cushing syndrome can be due to exogenous or endogenous causes. Nevertheless, appropriate differentiation of Cushing's disease and ectopic Cushing's syndrome is a major challenge for physicians due to their non-specific clinical symptoms and variable results of biochemical tests. From a therapeutic point of view this distinction is essential so that patients can be assuredly referred for the treatment of choice.

[0071] Pituitary microsurgery is one of the major tools to cure the Cushing's disease, for this an accurate localization of the tumour and preserving normal pituitary functions are essential requirements. Nowadays, contrast enhanced Magnetic Resonance Imaging (MRI) has been used for imaging studies and this technique has a sensitivity of 70%. Moreover, sometimes MRI scanning also gives false positives results in patients suffering from pituitary incidentaloma. Bilateral inferior petrosal sinus sampling (BIPSS) is an invasive, technically demanding test with limited availability.

[0072] The present work discloses a novel integrated functional plus anatomical imaging modality using radiolabelled DOTA conjugated modified Desmopressin (mDesmo) or .sup.68Ga-mDesmo PET/CT in the differential diagnosis and localization of ACTH dependent Cushing's syndrome. This is a novel modality and has not been used previously. One study used .sup.18F-Fluro-deoxy glucose (FDG)-PET/CT after intravenous administration of CRH (non-radioactive) and suggested that giving CRH prior to PET imaging improves the efficacy of .sup.18F-FDG for detecting pituitary tumor. However, .sup.18F-FDG is a nonspecific agent and the uptake of this is dependent on the metabolic activity of the tumor. It is taken up by all metabolic active tumors. Moreover, the interpretation of .sup.18F-FDG PET/CT is difficult in pituitary as the brain parenchyma has a bright physiological uptake of .sup.18F-FDG. .sup.68Ga-mDesmo is taken up specifically by corticotropinoma and not by brain parenchyma and normal pituitary. Therefore, .sup.68Ga-mDesmo PET/CT is a novel integrated functional plus anatomical imaging modality.