COMPOSITION FOR POSITRON EMITTING TOMOGRAPHY IMAGING IN CUSHING'S SYNDROME

TECHNICAL FIELD

10 [001] The present invention relates to development and use of radiopharmaceuti cal composition for PET imaging in patients with Cushing’s syndrome. More par ticularly the work provides a stable radiopharmaceutical composition comprising radioisotope, corticotrophin-releasing hormone (CRH) and bifunctional chelator- complex for imaging in adrenocorticotropin hormone (ACTH) dependent Cush- 15 ing’s syndrome. The invention relates to delineate corticotropinoma in patients with Cushing’s syndrome by employing this radiopharmaceutical composition.

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).

20 BACKGROUND ART

[002] Cushing’s syndrome is a clinical condition characterized by excessive circu lating cortisol and by various clinical features such as gain of weight, menstrual, disturbances, hirsutism, psychiatric dysfunction, proximal muscle weakness, os teoporosis, fractures, loss of scalp hair, plethora, rounding of face, hypertension,

25 easy bruising, wide purple striae, (>lcm) hyper pigmentation, and diabetes. Cush ing syndrome can be due to exogenous or endogenous causes.

[003] Exogenous Cushing's syndrome occurs due to excessive use of glucocorti coids whereas endogenous Cushing's syndrome is induced by inappropriate secre tion of cortisol owing to abnormalities in the hypothalamic pituitary adrenal 30 (HPA) axis. In normal physiology, hypothalamus produces CRH. This CRH binds to CRH receptors on the anterior pituitary gland leading to release of adrenocorti cotropic hormone (ACTH). Further, ACTH binds to receptors present on the ad renal cortex and stimulates the release of cortisol. Excessive production of ACTH from the pituitary gland or from ectopic source (non-pituitary) triggers the exces-

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SUBSTITUTE SHEETS (RULE 26) sive release of cortisol from adrenals resulting in Cushing’s syndrome. Cushing’s syndrome is diagnosed by biochemically documenting the presence of hypercoti- solism.

[004] After biochemically confirming the endogenous hypercortisolism, the next step is to determine ACTH dependency, which can be readily established by esti mating serum plasma ACTH level. Serum ACTH level more than 20 pg/ml sug gests ACTH dependent Cushing’s syndrome. ACTH dependent Cushing’s syn drome can be due to pituitary source or ectopic production of ACTH.

[005] In Endogenous ACTH-dependent Cushing’s syndrome, roughly 80% of pa tients suffers from Cushing’s disease caused by a pituitary adeno- ma(corticotropinoma), whereas 20% of total patients are of ECS caused by tu mour situated outside the pituitary gland.

[006] Nevertheless, inappropriate 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 with pituitary disease can be assuredly referred for the treatment of choice.

[007] Pituitary microsurgery is one of the major tools to cure the Cushing’s dis ease, for this an accurate localization of the tumour and preserving normal pitui tary 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 pos itives results in patients suffering from pituitary incidentaloma.

[008] Bilateral inferior petrosal sinus sampling (BIPSS) is an invasive, technically demanding test with limited availability. Intravenous administration of CRH is used during BIPSS to stimulate ACTH production.lt has sensitivity and specificity of more than 95% for differentiation of pituitary vs. ectopic production of ACTH However, lateralization accuracy that is the ability to lateralize the side of cortico-

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SUBSTITUTE SHEETS (RULE 26) tropinoma in the pituitary is only 70%, thereby limiting its utility to guide surgical procedure. Surgery done based on BIPSS results in care rate of 50-60%. BIPSS is an invasive procedure and is associated with potential serious complications, in cluding deep venous thrombosis, pulmonary emboli, and brain stem vascular damage. Since the procedure is technical and invasive, experienced teams will have better results and fewer complications. CRH used in BIPSS is generally safe but may be associated with mild side effects like tachycardia, hypotension, dizzi ness and flushing. Vasopressin causes numerous side effects such as abdominal pain or cramps, nausea, vomiting, pale skin, increased urge to bowl movements, dizziness, light headedness and hypertension. The present invention can not only differentiate pituitary vs. ectopic source of ACTH production but also is able to anatomically delineate the lesion and guide surgical therapy.

[009] The radiolabeling technique uses molecule (CRH) labeled with diagnostic radioisotopes (Gamma or Positron emitter) to observe uptake in pituitary by PET scanner. The invention involves the radiopharmaceutical composition comprises radioisotope, CRH and a bifunctional chelator, which has been further used to lo calize pituitary corticotropinoma.

DISCLOSURE OF THE INVENTION

[010] The present invention provides a radiopharmaceutical composition compris- ing CRH, a bifunctional chelator and further conjugated with a positron emitting radioisotope to image corticotropinoma in patients with Cushing’s syndrome. The complex along with PET/CT represent a novel functional imaging in patient with ACTH dependent Cushing's disease for tumour localization, especially in those cases wherein tumour is 1) not visualized with routine imaging, 2) localized to pituitary with a size <6 mm, 3) localized to pituitary with size > 6mm and dis cordant noninvasive dynamic testing. In these cases, it may obviate the need for BIPSS which is limited by many factors and associated adverse events. This com-

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SUBSTITUTE SHEETS (RULE 26) bined imaging modality like PET/CT or PET/ MRI may further enhance its diag nostic capabilities.

BRIEF DESCRIPTION OF TABLES AND DRAWINGS [Oil] The present invention will become more understandable from the descrip tion given herein and the accompanying drawings below. These are given by way of illustration only and therefore not limited to present invention and wherein:

Table 1: Results of Ga-68 CRH PET-CT in patients with ACTH dependent Cush ing's Table 2. Diagnostic accuracy of Ga-68 CRH, MRI, Inferior petrosal sinus sam pling and High dose dexamethasone suppression test in diagnosis of ACTH de pendent Cushing's

Figure 1: The HPLC chromatogram (A) and mass spectra (B) of DOTA-CRH. The HPLC was done using water and acetonitrile as mobile phase. The retention time of DOTA-CRH was observed to be 10.9 min.

Figure 2: Radio-ITLC chromatogram of Ga-68-DOTA-CRH using sodium citrate as mobile phase. Only single peak was observed at the origin indicating absence of any free Ga-68 (R _f 1.0). The R _f of Ga-68-DOTA-CRH was observed to be 0.0. Figure 3: A 63 years old male (Patientl) with complaint of headache and disturb- ance in vison. MRI showed localized mass in sella and underwent serial brain spots and whole-body Ga-68-DOTA-CRH PET/CT imaging. Tracer uptake was noted in lesion on serial brain PET image acquisition with SUVmax 5.1 (5 min), SUVmax 6.3 (10 min), SUVmax 5.48 (15 min), SUVmax 6.29 (20 min), SUVmax 5.05 (25 min), SUVmax 6.8 (30 min), SUVmax 6.8 (35 min), SUVmax 5.14 (40 min), and SUVmax 6.8 (45 min) at 300 s, post 3.12 mCi of Ga-68-DOTA-CRH administration.

Figure 4: Whole body image of 63 years old male (Patientl) acquired 45 min post administration (90 s/frame) of Ga-68-DOTA-CRH. Maximum intensity projection

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SUBSTITUTE SHEETS (RULE 26) image (MIP) shows physiological uptake of Ga-68-DOTA-CRH in liver and spleen with diffused uptake in bone marrow. The excretion was observed via kid neys into urinary bladder. Focal uptake of radiotracer was noted in midline in the head region, corresponding to lesion in pituitary (SUVmax 4.8) in transaxial CT (B) and fused PET/CT (C) images.

Figure 5. 31 year old male with recurrence of Cushing's disease seven years after successful TSS for macroadenoma. Coronal T2W (A) and gadolinium enhanced T1W MRI images (B) showing post-operative changes. (C) Ga-68 CRH PET-CT showing focal uptake in right half of pituitary.

BEST MODE(S) FOR CARRYING OUT THE INVENTION [012] The following description describes various features and functions of the disclosed composition and method. The illustrative composition and method de scribed herein are not meant to be limiting. It can be readily understood that cer tain aspects of the disclosed composition and method can be arranged and com bined in a wide variety of different configurations, all of which are contemplated herein.

[013] 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. 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. 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 ap parent to those skilled in the art that the following description of exemplary em bodiments 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.

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SUBSTITUTE SHEETS (RULE 26) [014] It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[015] By the term “substantially” it is meant that the recited characteristic, pa rameter, or value need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement error, measurement accuracy lim itations 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.

[016] 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.

[017] The term “radiolabeling” disclosed herein refers to the binding of radionu clide with molecule of interest such as peptide.

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

[019] The term “PET (Positron Emitting Tomography)” disclosed herein re fers to the nuclear medicine tomographic imaging technique using positrons.

[020] The term “CRH” disclosed herein refers to the synthetic hormone which binds to CRH receptors on corticotropes and stimulates the production of ACTH (Adrenocorticotropic Hormone).

[021] 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 or gans and tissues within the body.

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SUBSTITUTE SHEETS (RULE 26) [022] 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

[023] The term “corticotropinoma” disclosed herein refers to the pituitary ade- noma made up predominantly of corticotrophs.

[024] The term “bifunctional chelator” disclosed herein refers to the molecule that has ability to bind with biomolecule and has metal chelation property.

[025] The term “differential diagnosis” disclosed herein refers to distinguish be tween Cushing's disease and ectopic Cushing’s syndrome (ECS). [026] CRH, a 41 amino acid sequence (S EEPPIS LDLTFHLLRE VLEM A-

RAEQLAQQAHSNRKLMEII), was custom synthesized and conjugated with DOTA. Firstly conjugated with a bifunctional chelator followed by purification with high-performance liquid chromatography (HPLC). For HPLC, water (A) and acetonitrile (B) with 0.1% trifluoroacetic acid (TFA) is used as the mobile phase. Subsequently the said complex is further labelled with a radioisotope.

EMBODIMENTS

[027] In an embodiment CRH, a 41 amino acid sequence, is conjugated with a bi functional chelator selected from a group comprising of NOTA, NODAGA, DOTAGA, DTPA, DOTA, D03A, NOTA, NOTADA, DOTAGA, NODAGA, DOTA-dPEG, DTPA, DOTMP, DOTPA, DOTAZA, DOTG, TETA, TETPA, PCTA, NETA, NE3TA, DE4TA, DEPA, CNETA, CNEJTA, HYNIC, OXIME, TROPONINE, DFO, DCPA,MACROPA,PEPA, PCTA, TRAP, N02AP, NS3,HEHA, EDTA, DMSA, N2S2,N3S, HBED-CC followed by purification with high-performance liquid chromatography (HPLC).

[028] In an embodiment CRH, a 41 amino acid sequence, is conjugated with at- least one bifunctional chelator selected from a group comprising of derivatives of

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SUBSTITUTE SHEETS (RULE 26) NOTA, NODAGA, DOTAGA, DTPA, DOTA, D03A, NOTA, NOTADA, DOTAGA, NODAGA, DOTA-dPEG, DTPA, DOTMP, DOTPA, DOTAZA, DOTG, TETA, TETPA, PCTA, NETA, NE3TA, DE4TA, DEPA, CNETA, CNEJTA, HYNIC, OXIME, TROPONINE, DFO, DCPA,MACROPA,PEPA, PCTA, TRAP, N02AP, NS3,HEHA, EDTA, DMSA, N2S2,N3S, HBED-CC fol lowed by purification with high-performance liquid chromatography (HPLC).

[029] In a preferred embodiment the bifunctional chelator is DOTA (1,4,7,10- tetraazacyclododec ane- 1 ,4 ,7 , 10-tetraacetic acid) .

[030] CRH (lmg) is dissolved DMF in sodium bicarbonate buffer (pH-9). DOTA in sodium bicarbonate buffer is added to peptide solution at molar ratio of 1:1 to 1:5 (peptide to DOTA), mixed well and incubated at 4° C for 24 hr. The conjugat ed peptide was purified using size exclusion chromatography and characterized by HPLC and mass spectra. For HPLC, acetonitrile and water were used as mobile phase with 0.1% trifluoroacetic acid (TFA). Peptide was lyophilized at -20°C till further use.

[031] Referring to Figure 1: In an embodiment for HPLC, water and acetonitrile with 0.1% trifluoroacetic acid (TFA) is used as the mobile phase (Fig 1A). the mass of purified conjugated CRH is verified by Electrospray ionization mass spectra (ESI-MS) (Fig IB).

[032] In an embodiment, the number of conjugated DOTA molecules are deter mined by subtracting the mass of unconjugated CRH from conjugated CRH and dividing with the mass of DOTA. The mass of DOTA-CRH, CRH and DOTA are 5144, 4757 and 688 daltons.

[033] In an embodiment in the complex CRH peptide, bifunctional chelator and radioisotope are in any molar ratio.

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SUBSTITUTE SHEETS (RULE 26) [034] In a preferred embodiment in the complex CRH peptide, bifunctional chela tor and radioisotope are in 1:1 molar ratio.

[035] In an embodiment CRH-bifunctional chelator complex is further conjugated with atleast one isotope selected from the group consisting of phosphorus, calci um, sodium, rhenium, scandium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, actinium-225, antimony- 127, arsenic-74, barium-140, bismuth-210, califomium-246, calcium- 46, calcium-47, carbon- 11, carbon-14, cesium- 131, cesium-137, chromium-51, cobalt-57, cobalt-58, cobalt- 60, dysprosium- 165, erbium- 169, fluorine-18, gallium-67, gallium-68, gold-198, holmium- 166, hydrogen-3, indium-111, indium-113m, iodine-123, iodine- 125, iodine- 131, iridium-192, iron-59, iron-82, krypton-81m, lanthanum- 140, luteti- um-177, molybdenum-99, nitrogen-13, oxygen-15, palladium- 103, phosphorus- 32, radon-222, radium-224, rhenium- 186, rhenium- 188, rhodium-82, samarium- 153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thalli um-201, xenon-127, xenon-133 and yttrium-90.

[036] In a preferred embodiment CRH-bifunctional chelator complex is further conjugated with isotope Ga-68.

[037] Radiolabeling of DOTA-CRH with Ga-68: Lyophilized DOTA-CRH was dissolved in water (1 mg/mL) and thereafter stored at -20°C. Radiolabeling of DOTA-CRH (20 pg) was done with Ga-68 freshly eluted from Ge-68/Ga-68 gen erator at 95 °C for 10 min (acidic pH). The purification of radiolabeled products, Ga-68 DOTA-CRH or Ga-68 CRH, was done using preconditioned C-18 car tridge. The characterization of purified Ga-68-CRH was done by MALDI-TOF and HPLC with radioactivity detector and the retention time of the product from the column was recorded.

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SUBSTITUTE SHEETS (RULE 26) [038] In an embodiment bifunctional chelator and radioisotope are DOT A and Ge-68 respectively.

[039] Referring to Figure 2: Radio-ITLC chromatogram of Ga-68-DOTA-CRH using sodium citrate as mobile phase. Only single peak was observed at the origin indicating absence of any free Ga-68 (R _f 1.0). The R _f of Ga-68-DOTA-CRH was observed to be 0.0.

[040] Quality control of Ga-68 CRH: Quality control tests such as radiolabeling efficiency and radiochemical purity of the labelled product were determined by thin-layer chromatography (TLC) on silica gel-coated strips using 0.5 M sodium citrate solution (pH 5.5) and NH ₄ 0Ac:MeOH (1:1) solution as the mobile phases. Sterility testing was done by incubating the radiolabeled sample volume equiva lent to the injection volume (2 mL) in tryptic soya broth medium (10 mL) at 37°C. Turbidity in broth was observed up to 7 d for the presence of any growth.

Pyrogen testing was done using a point-of-use portable test system. To determine the endotoxin contents, 25 pL of the sample was added to each well in the PTS cassette and was incubated at 38°C for 15-20 min. The observations were noted with 50% -200% spike recovery with sensitivity of 0.01 endotoxin unit (EU)/ml.

Gas chromatography was used to measure the residual ethanol content in the product. Based on area under the curve of ethanol standard (4000 ppm), the con centration of ethanol in the product was calculated. The stability of both com pounds was checked by incubating the radiolabeled samples with phosphate- buffered saline (PBS) at room temperature for up to 6 h.

Patient Imaging:

Referring to Figure 3, Acquisition of Serial PET images up to 45 min followed by whole-body PET/CT imaging. Based on the mean SUVmax obtained from serial images, the optimum imaging time was observed to be 35-40 min post administration of 3-5 mCi of Ga-68-DOTA-CRH.

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SUBSTITUTE SHEETS (RULE 26) Referring to Figure 4, the physiological distribution of Ga-68-DOTA-CRH was noted in the liver and spleen with diffused uptake in bone marrow and pituitary. Excretion of Ga-68 CRH was observed mainly via the renal route.

[041] Clinical studies in patients with ACTH dependent Cushing's syndrome: In subjects with clinical suspicion of Cushing's syndrome, exogenous steroid use was ruled out by using 0800h cortisol. Subsequent screening for Cushing's was done using overnight dexamethasone suppression test (ONDST) and 2300h corti sol. ONDST was performed by administration of 1 mg of oral dexamethasone at 2300h and sample for cortisol was taken the next day at 0800h, values > 50 nmol/1 were considered as non-suppressible. Sampling for late night cortisol was done in an awake state at 2300h and values > 207 nmol/1 were considered as evident of loss of circadian rhythm. In subjects with either elevated late-night cortisol or non-suppressible ONDST, low dose dexamethasone suppression (LDDST) was done as a confirmatory test. LDDST was performed by administration of 0.5 mg oral dexamethasone every 6 hours for 48 hours and cortisol sample was with drawn at 0800h on the third day. Cortisol value > 50 nmol was considered signifi cant. In biochemically confirmed Cushing's ACTH.

METHOD OF DIFFERENTIAL DIAGNOSIS OF CUSHING'S SYN- DROME

[042] All those patients with confirmed diagnosis of ACTH dependent Cushing's syndrome were subjected to Dynamic CEMRI of sella and 3D MP RAGE T1 weighted imaging on 3T MRI machine were taken. In selected cases high dose dexamethasone suppression test (HDDST), and/or inferior petrosal sinus sampling (IPSS) were done, which was decided on an individual basis by the treating team. HDDST was performed by administration of 2 mg oral dexamethasone every 6 hours for 48 hours and 0800h cortisol sample was withdrawn on day 3. Cortisol suppression of more than 50% from baseline after HDDST was considered as suggestive of Cushing's disease.

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SUBSTITUTE SHEETS (RULE 26) [043] Material and Methods: It was a single centre prospective study carried out in the Department of Endocrinology, Post Graduate Institute of Medical Educa tion and Research, Chandigarh. In subjects with clinical suspicion of Cushing's syndrome, exogenous steroid use was ruled out by using a 0800hr cortisol value of > 50 nmol/1. Subsequent screening for Cushing's was done using 2300hr corti sol and overnight dexamethasone suppression test (ONDST). Sampling for late night cortisol was done in a awake state at 2300hr and value > 207 nmol/1 was considered as evident of loss of circadian rhythm. ONDST was performed by ad ministration of 1 mg of oral dexamethasone at 2300hr and sample for cortisol was drawn the next day at 0900 hr, values > 50 nmol/1 were considered as non- suppressible. In subjects with elevated 2300hr cortisol and/or non-suppressible ONDST, low dose dexamethasone suppression (LDDST) was done as a confirma tory test. LDDST was performed by administration of 0.5 mg oral dexamethasone every 6 hours for 48 hours and fasting cortisol sample was withdrawn 6 hours af ter the last dose, value > 50 nmol/1 were considered significant. In patients having Cushing's syndrome, ACTH dependency was determined by 0800hr ACTH value of > 20 pg/ml.(7) All those with biochemically confirmed ACTH dependent Cushing's syndrome were subjected to dynamic CEMRI of sella and 3D MP RAGE T1 weighted imaging on 3T MRI machine. In selected cases high dose dexamethasone suppression test (HDDST) and/or bilateral inferior petrosal sinus sampling (BIPSS) were done, which was decided on an individual basis by the treating team. HDDST was performed by administration of 2 mg oral dexame thasone every 6 hours for 48 hours and fasting cortisol sample was withdrawn at 0800hr on day 3, cortisol suppression of more than 50% from baseline was con sidered as suggestive of Cushing's disease. BIPSS was performed as per the insti tute protocol, details of which have been published elsewhere. (3) After obtaining ethical clearance from institutional ethics committee(NK/37771/Study/544) and informed consent, Ga-68 CRH PET/CT scan was performed in subjects with ACTH dependent Cushing's syndrome.

[044] The CRH was custom synthesized and DOTA was conjugated to it. HPLC purified (>95%) DOTA conjugated CRH was lyophilized and was used for radio

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SUBSTITUTE SHEETS (RULE 26) labeling of DQTA CRH with Ga-68. Whole body and static (head) images were acquired after 45-60 min post intravenous injection of 111-185 MBq Ga-68 CRH on a dedicated hybrid PET-CT scanner (Discover 710, GE Healthcare, Milwau kee, USA). CT acquisition was performed having CT parameters 120 kV, 350 mA with rotation time of 0.5 sec and slice thickness of 3.75 mm, 512 x 512 pixels ma trix and pixel size of about 1mm were used. After CT acquisition, 3 D PET acqui sition was done using 128x128 pixels matrix with slice thickness of 3.25 mm. CT based attenuation correction was used for PET images. PET and CT images were reconstructed by using Iterative method ordered subset expectation maximization (OSEM) and filter back projection (FBP), respectively. Images such acquired were superimposed on MRI images andinterpreted by a panel of two nuclear med icine experts, who were blinded for the biochemical and otherradiological imag ing results.

[045]-The diagnosis of Cushing’s disease was considered established if:

1) Histological examination of a surgical specimen confirmed it (n=20), or

2) transsphenoidal surgery resulted in resulted in hypocortisolism or eucortisolic state and clinical remission even though no tumour tissue was identified on histo- pathology(n=l), or

3) in cases of ACTH dependent Cushing's syndrome a suppressible HDDST, MRI showing pituitary adenoma and a negative DOT ATE whole body PET-CT and CECT chest plus abdomen (n=l).

This patient underwent bilateral adrenalectomy due to significant co morbidities making the patient unfit for trans-sphenoidal surgery. The diagnosis of ectopic Cushing’s syndrome was based on surgical excision and histopathology of the primary tumour responsible for ACTH/CRH production (n=2).

[046] Hormonal analysis: Serum cortisol and ACTH were measured by electro- chemiluminescence-immuno-assay (ECLIA) (ELECSYS-2010, Roche Diagnos tics, Germany). The range of ACTH measurement was 1.5 to 2000 pg/ml with limit of detection and quantitation of 1.5 and 3 pg/ml, respectively. The range of

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SUBSTITUTE SHEETS (RULE 26) cortisol measurement was 1.5 to 1750 nmol/1 with limit of detection and-quantita- tion of 1.5 and 3.0 nmol/1, respectively.

[047] Statistical Analysis: Discrete categorical variables were presented as n(%); continuous data were given as mean ± SD & range or median and inter quartile range, as appropriate. Investigations were categorised into the categories nor- mal/abnormal according to standard cut-off values. Analysis was conducted using SPSS for windows (version 21.0; SPSS Inc, Chicago, IL, USA).

[048] RESULTS

Table 1: Diagnosis of patients with ACTH dependent Cushing's who underwent Ga-68 CRH PET CT

A total of 24 patients with ACTH dependent Cushing's syndrome underwent Ga- 68 CRH PET/CT, out of which 22 were having Cushing's disease and two had ec topic ACTH syndrome. In all the cases of Cushing's disease, diagnosis was based on the histopathology of the excised tissue confirming corticotropinoma except in two patients. Both had pituitary adenoma on CEMRI, while DOTATE PET-CT and CECT chest plus abdomen did not reveal any ectopic source. One patient un derwent bilateral adrenalectomy due to associated severe co-morbidities, and in the other excised tissue did not reveal adenoma(failed surgery). The aetiology of ectopic Cushing's in both the cases was bronchial carcinoid and was confirmed on HPE and documentation of remission after primary tumour excision. Of 24 cases 16 were females with a median age of 35.5 years and range of 13 to 64 years.

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SUBSTITUTE SHEETS (RULE 26) Table 2. Diagnostic accuracy of Ga-68 CRH, MRI, Inferior petrosal sinus sam pling and High dose dexamethasone suppression test in diagnosis of ACTH de pendent Cushing's of corticotropinoma in the sella, NA: not applicable Of the 22 cases with Cushing's disease Ga-68 CRH scan was able to localize the source in pituitary in all except one. In this case ACTH was less than 90 pg/ml, HDDST was suppressible, MRI revealed a doubtful pituitary adenoma of size 3 x 2 mm and DOTATE PET-CT and CECT chest plus abdomen did not reveal any ectopic source. The patient couldn't go under BIPSS in view of financial con- straints and subjected to total bilateral adrenalectomy with histopathology show ing adrenal hyperplasia and is under monitoring for development of nelson syn drome

[049] Among the two cases with ectopic ACTH syndrome, Ga-68 CRH PET-CT scan showed uptake in one which was diffuse while there was no uptake in the

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SUBSTITUTE SHEETS (RULE 26) other patient. The subject with diffuse uptake on CRH PET/CT had a central gra dient on BIPSS while MRI revealed a pituitary microadenoma. Interestingly, in view of clinical presentation and diffuse uptake on CRH PET CT, patient was subjected to Ga-68 DOTATE PET-CT which revealed a lesion in right lung upper lobe. Subsequently, patient was subjected to bilateral adrenalectomy due to pres- ence of co-morbid conditions and one year later after optimization, right lobecto my which revealed bronchial carcinoid on histopathology. The result of BIPSS and CRH PET were consonant with the CRH producing ectopic tumour resulting in corticotroph hyperplasia and central gradient of ACTH. Moreover, repeat MRI revealed subsidence of the hyperplasia and regression of adenoma after removal of the ectopic tumour.

Table3. Results of Ga-68 CRH PET-CT in patients with ACTH dependent Cush ing's

DISCUSSION [050] Ga-68 CRH PET-CT is based on the premise of binding of Ga-68 CRH to

CRH receptors on corticotropinoma. Hence it not only delineates the adenoma, but also confirms its functionality making it an integrated functional plus anatom ical imaging. In our study, it was able to correctly localize the lesion in 21 out of 22 cases including 16 cases with micro corticotropinoma. More importantly, it could identify the culprit lesion in 8 out of 9 cases with tumour size less than 6 mm including two cases where MRI was normal. It also correctly lateralizes the lesion in 15 of the cases with microadenoma and had a better performance than

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SUBSTITUTE SHEETS (RULE 26) either MRI or BIPSS in this regard. There was no uptake in pituitary in one pa tient with ectopic ACTH syndrome (Figure 5), while it revealed a diffuse uptake in the other with CRH producing bronchial carcinoid. Interestingly, in this case MRI revealed a pituitary microadenoma and the presence of diffuse uptake on CRH lead to a decision of Ga-68 DOTATE PET-CT which showed a right lung upper lobe bronchial carcinoid , hence an alteration in diagnosis and subsequently curative resection of bronchial carcinoid.

GA-68 CRH PET-CT V/S OTHER NUCLEAR MEDICINE IMAGING MODALITIES

[051] Nuclear medicine imaging modalities have gained an increasing role in evaluation of ACTH dependent Cushing's. For delineation of corticotropinoma F- 18 FDG and C-ll methionine PET-CT have been employed, albeit with a limited success. In a prospective study of ten consecutive patients with Cushing's disease, high resolution F-18 FDG-PET revealed a tumour in 4 as compared to 7 with SPGR MRI sequence, while in another C-ll methionine PET-CT was non diag nostic in 30%. Moreover, the premise of both of these imaging i.e. increased glu cose metabolism and protein synthesis is not unique to corticotropinoma and is rather low. Moreover, the surrounding brain parenchyma has high metabolic ac tivity making visualization of corticotropinoma difficult. On the other hand, Ga- 68 CRH PET-CT is based on of binding of Ga-68 CRH to CRH receptors on cor ticotropinoma which are up regulated. Hence as compared to other nuclear imag ing modalities, Ga-68 CRH PET-CT is not only more sensitive but also confirms its functionality making it a true integrated functional plus anatomical imaging, with a success rate of 91.6% in the present study.

[052] The present study has for the first time used a novel imaging modality for corticotropinoma which integrates functionality of the tumour with anatomical localization. Moreover, diagnosis was established by histopathological evaluation in majority (22/24) of the cases.

[053] The processes described above is described as a sequence of steps, this was done solely for the sake of illustration. Accordingly, it is contemplated that some

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SUBSTITUTE SHEETS (RULE 26) 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.

[054] Although embodiments have been described with reference to specific ex ample 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.

[055] 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 fore going description. It is to be understood that the phraseology or terminology em ployed herein is for the purpose of description and not of limitation. It is to be un derstood that the description above contains many specifications; these should not be construed as limiting the scope of the invention but as merely providing illus trations of some of the personally preferred embodiments of this invention.

INDUSTRIAL APPLICABILITY

[056] The present invention is to provide a pharmaceutical composition compris ing conjugation of corticotrophin-releasing hormone (CRH) peptide and a bifunc tional chelator for the differentiational diagnosis of Cushing’s syndrome and de lineation of the corticotropinoma, wherein CRH is conjugated with a bifunctional chelator to facilitate radiolabelling with radioisotope.

[057] The present invention is to provide a radiolabeling method, wherein radio labeling has been performed with radioisotope which act as tracer for Positron Emission Tomography (PET).

[058] The complex along with PET/CT represent a novel functional imaging in patient with ACTH dependent Cushing's disease for tumour localization, especial ly in those cases wherein tumour is 1) not visualized with routine imaging, 2) lo calized to pituitary with a size <6 mm, 3) localized to pituitary with size > 6mm and discordant noninvasive dynamic testing. In these cases, it may obviate the

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SUBSTITUTE SHEETS (RULE 26) need for BIPSS which is limited by many factors and associated adverse events. This combined imaging modality like PET/CT or PET/ MRI may further enhance its diagnostic capabilities.

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SUBSTITUTE SHEETS (RULE 26)