J Radioanal Nucl Chem (2014) 299:1389–1398
`DOI 10.1007/s10967-013-2894-z
`
`Preparation of DOTA-TATE and DOTA-NOC freeze-dried kits
`for formulation of patient doses of 177Lu-labeled agents and their
`comparison for peptide receptor radionuclide therapy application
`
`Tapas Das • Mohini Bhadwal • Sharmila Banerjee •
`H. D. Sarma • Ajit Shinto • K. K. Kamaleshwaran
`
`Received: 8 August 2013 / Published online: 4 January 2014
`Ó Akade´miai Kiado´, Budapest, Hungary 2014
`
`Abstract The objective of the present work is to prepare
`freeze-dried DOTA-TATE and DOTA-NOC kits for the easy
`and convenient preparation of patient doses of 177Lu-DOTA-
`TATE and 177Lu-DOTA-NOC, respectively at the hospital
`radiopharmacy and to compare the radio-peptides with respect
`to their radiochemical and biological behaviors. Freeze-dried
`kits of DOTA-TATE and DOTA-NOC, comprising a lyoph-
`ilized mixture of 200 lg of DOTA-peptide, 80 mg of gentisic
`acid and 13.9 mg of ammonium acetate were prepared.
`Therapeutic doses of 177Lu-labeled peptides (up to 200 mCi,
`7.4 GBq) were prepared using these kits and 177Lu, produced
`in-house, with[99 % radiochemical purity and high stability
`following an easy and convenient protocol. Comparative
`pharmacokinetic behavior of the radio-peptides was studied
`by carrying out biodistribution studies in normal Wistar rats
`
`T. Das (&) M. Bhadwal S. Banerjee
`Isotope Applications and Radiopharmaceuticals Division,
`Bhabha Atomic Research Centre, Trombay, Mumbai 400 085,
`India
`e-mail: tdas@barc.gov.in
`
`M. Bhadwal
`e-mail: mohini@barc.gov.in
`
`S. Banerjee
`e-mail: sharmila@barc.gov.in
`
`H. D. Sarma
`Radiation Biology and Health Sciences Division, Bhabha
`Atomic Research Centre, Trombay, Mumbai 400 085, India
`e-mail: hdsarma@barc.gov.in
`A. Shinto K. K. Kamaleshwaran
`Department of Nuclear Medicine & PET, Kovai Medical Centre
`and Hospital, Coimbatore 641 014, Tamil Nadu, India
`e-mail: ajitshinto@gmail.com
`
`K. K. Kamaleshwaran
`e-mail: dr.kamaleshwar@gmail.com
`
`which revealed higher retention of activity in several major
`organs and slower renal clearance for 177Lu-DOTA-NOC
`compared to that of 177Lu-DOTA-TATE. Preliminary phar-
`macokinetic studies, carried out in limited number of patients
`suffering from cancers of neuroendocrine origins, showed
`lower accumulation of activity in vital organs and faster
`renal clearance of 177Lu-DOTA-TATE compared to that of
`177Lu-DOTA-NOC.
`Keywords Freeze-dried DOTA-TATE kit
`Freeze-dried DOTA-NOC kit 177Lu-DOTA-TATE
`177Lu-DOTA-NOC PRRNT Neuroendocrine cancers
`
`Introduction
`
`Peptide receptor radionuclide therapy (PRRNT) employing
`radiolabeled somatostatin analogue peptides has become an
`established procedure for the treatment of patients suffer-
`ing from inoperable neuroendocrine cancers over-express-
`ing somatostatin receptors (SSTR) in the last decade [1–6].
`The use of several radiolabeled peptides such as, 90Y-
`DOTA-TATE
`(1,4,7,10-tetraazacyclododecane-1,4,7,10-
`tetraacetic acid coupled Tyr3-octreotate), 90Y-DOTA-TOC
`177Lu-DOTA-TATE,
`(DOTA coupled Tyr3-octreotide),
`177Lu-DOTA-TOC and 177Lu-DOTA-NOC (DOTA cou-
`pled Nal3-octreotide) have been documented for this pur-
`pose [1–11]. Amongst the 177Lu-labeled peptides, 177Lu-
`DOTA-TATE and 177Lu-DOTA-TOC have been used
`more frequently for treating the neuroendocrine cancer
`patients while only limited use of 177Lu-DOTA-NOC have
`been reported [12]. In India, 177Lu-DOTA-TATE is regu-
`larly used in several nuclear medicine centers for providing
`radiotherapeutic treatment to the patients suffering from
`various types of inoperable neuroendocrine cancers [13,
`
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`14]. However, recent non-availability of DOTA-TATE and
`DOTA-TOC due to intellectual property right issues has
`created inconvenience to the patients who are undergoing
`radiotherapeutic regimen as well as for the new patients
`who are scheduled to undergo PRRNT using 177Lu-DOTA-
`TATE. This necessitated studying other DOTA coupled
`somatostatin analog peptides for labeling with 177Lu. The
`availability of DOTA-NOC coupled with the fact that it has
`the highest affinity towards SSTR-3 and SSTR-5 and high
`affinity towards SSTR-2 among all the somatostatin ana-
`logs considered [12, 15, 16], has prompted us to radiolabel
`it with 177Lu and also to carry out a comparative study
`between 177Lu-DOTA-NOC and 177Lu-DOTA-TATE in
`biological systems.
`The success of PRRNT, along with several other factors,
`primarily depends on the availability of the radiolabeled
`peptide with adequately high specific activity, so that
`required therapeutic efficacy can be achieved without satu-
`rating the limited number of receptors available on the target
`lesions [17–19]. This in turn directly depends on the specific
`activity of the radionuclide at the time of preparation of the
`agent. Therefore, 177Lu-DOTA-TATE, the agent which is
`most commonly used in India for treating neuroendocrine
`cancer patients, is prepared in the hospital radiopharmacy
`just prior to its administration in patients [13, 14, 20]. This
`ensures preparation of the agent with maximum possible
`therapeutic efficacy depending on the specific activity of
`177Lu available at the time of preparation. However, suc-
`cessful preparation of the agent following this methodology
`is largely dependent on the availability of the trained per-
`sonnel at the respective nuclear medicine centers, as this
`procedure requires stringent adjustment of certain parame-
`ters prior to incubation [20]. A small deviation from the
`standard procedure may lead to failure of the batch and
`consequently loss of expensive peptide and radionuclide.
`Moreover, this may adversely affect the treatment schedule
`of the patients. However, all these problems can be cir-
`cumvented if the freeze-dried kits, which will enable the
`preparation of the radiolabeled peptide in an easy and single-
`step, could be developed. The use of kits is also expected to
`reduce the exposure of the personnel working in the hospital
`radiopharmacy as well as number of batch failures.
`Working in this direction, we have developed freeze-dried
`DOTA-NOC kits which can be utilized to prepare thera-
`peutic doses of 177Lu-DOTA-NOC using 177Lu having a
`certain minimum specific activity at the time of preparation
`of the agent. A similar attempt was also made to prepare
`freeze-dried DOTA-TATE kits, suitable for the preparation
`of patient doses of 177Lu-DOTA-TATE, in order to make a
`comparison between these two agents. It is worthwhile to
`mention that the use of freeze-dried DOTA-TATE kits for
`the formulation of 177Lu-DOTA-TATE have already been
`documented in the literature [21, 22]. However, none of these
`
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`
`articles describe the detailed methodologies of formulation of
`such kits and merely mention their use for the formulation of
`patient dose of 177Lu-DOTA-TATE. Moreover, no information
`is available regarding the rationale behind taking certain amount
`of DOTA-TATE, shelf-life of the kits and most importantly,
`about the maximum dose that can be prepared using a single kit
`vial. Apart from this, none of these kits have been utilized for
`the preparation of usually administrated therapeutic dose of
`5.55–7.4 GBq (150–200 mCi) 177Lu-DOTA-TATE.
`In the present paper, we describe the detailed method-
`ologies of preparation of freeze-dried DOTA-TATE and
`DOTA-NOC kits, whose one kit vial is sufficient for the
`preparation of up to 7.4 GBq (200 mCi) patient dose of
`177Lu-DOTA-TATE and 177Lu-DOTA-NOC, respectively.
`Herein we also report a comparative study of 177Lu-
`DOTA-TATE and 177Lu-DOTA-NOC, prepared using the
`respective kits, in terms of their radiochemical and bio-
`logical behaviors in animal models. Our experience of
`using these kits to treat limited number of neuroendocrine
`cancer patients is also documented in the present paper.
`
`Materials and methods
`
`DOTA-TATE acetate and DOTA-NOC acetate were obtained
`from ABX Advanced Biochemical Compounds (Germany).
`Gentisic acid (2,5-dihydroxybenzoic acid) and ammonium
`acetate were procured from Aldrich Chemical Company
`(USA). Lutetium oxide (82 % enriched in 176Lu, spectro-
`scopic grade,[99.999 % chemically pure) was obtained from
`Centre for Molecular Research (Russia). High purity supra-
`pure water and supra-pure HCl were obtained from Merck
`(Germany). All other chemicals and solvents used were of
`analytical reagent (AR) grade and supplied by reputed local
`chemical manufacturers. Radionuclidic purity of 177Lu was
`ascertained by high resolution gamma ray spectrometry using
`a HPGe detector (EGG Ortec/Canberra detector, USA) cou-
`pled to a 4K multichannel analyzer (MCA) system after
`radiochemical processing. All other radioactivity measure-
`ments were carried out by using well-type NaI(Tl) scintillation
`counter (Electronic Corporation of India Limited, India),
`unless mentioned otherwise, by keeping the baseline and
`window at 150 and 100 keV, respectively; thereby utilizing
`the 208 keV gamma photon of 177Lu. Lyophilization was
`done by using the Labocene CoolsafeTM 55-4 freeze-drier
`(Denmark). Paper chromatography (PC) strips were pur-
`chased from Whatman (UK). The high performance liquid
`chromatography (HPLC) system (PU 1580) was obtained
`from Jasco (Japan). The elution profile was monitored by
`detecting the associated radioactivity signal using a well-type
`NaI(Tl) detector (Jasco, Japan) coupled with the HPLC sys-
`tem. All the solvents used for HPLC were degassed and fil-
`tered prior to use and were of HPLC grade.
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`Animal experimentations were carried out in normal
`Wistar rats which were bred and reared in the laboratory
`animal facility of our Institute under standard management
`practice. Radioactive counting associated with the animal
`studies were carried out using a flat-type NaI(Tl) scintil-
`lation counter (Electronics Corporation of India Limited,
`India) fixing the baseline at 150 keV and keeping a win-
`dow of 100 keV. All the animal experiments were carried
`out in strict compliance with the relevant national laws
`relating to the conduct of animal experimentation.
`Clinical studies with 177Lu-DOTA-TATE and 177Lu-
`DOTA-NOC, prepared using the corresponding freeze-
`dried kits, were carried out by administering the prepara-
`tion to the patients suffering from inoperable or metasta-
`sized cancers of neuroendocrine origin. The patients
`showed somatostatin receptor positive disease in somato-
`statin receptor scintigraphy (SRS) carried out with 99mTc-
`HYNIC-TOC (hydroxynicotinamide coupled Tyr3-octreo-
`tide), prepared using in-house HYNIC-TOC kit, one month
`prior to the therapy. All the patients exhibited significantly
`higher uptake of activity in the tumors and metastatic
`lesions compared to that in liver in SRS study. Aminoven
`10 % intra-venous infusion, an injectible solution of the
`mixed amino acids, was obtained from Fresenius Kabi
`(Austria). Post-therapy whole-body scans and SPECT-CT
`(SPECT: single photon emission computed tomography,
`CT: computed tomography) acquisitions of the abdomen
`area were performed using the dual-head gamma camera
`(Symbia T-200) procured from Siemens (Germany). The
`necessary ethical clearances for administration of
`the
`agents in human patients were obtained from the competent
`authority and written consents were collected from the
`patients prior to the administration of the agents.
`
`Experimental
`
`Formulation of freeze-dried DOTA-TATE and DOTA-
`NOC kits
`
`Freeze-dried DOTA-TATE and DOTA-NOC kits (ten
`numbers in each batch) were prepared following the protocol
`mentioned below. A solution of gentisic acid in buffer was
`prepared by dissolving 800 mg of gentisic acid in 18 mL of
`0.1 M ammonium acetate buffer (pH 5) by gentle warming
`under aseptic conditions. A solution of the peptide, prepared
`by dissolving 2 mg of the DOTA-TATE or DOTA-NOC in
`2 mL of high purity supra-pure water, was added with the
`buffer solution containing gentisic acid. The resultant solu-
`tion was thoroughly mixed and its pH was adjusted to *5.
`Ò
`The solution was subsequently passed through Millipore
`(0.22 l) filter paper and aliquoted into ten sterile glass vials,
`each vial containing 2 mL of the solution. All
`these
`
`preparative steps were carried out under aseptic conditions.
`The vials were incubated for a period of 24 h at -4 °C fol-
`lowed by another 24 h at -40 °C. Finally, the vials were
`freeze-dried in a lyophilizer for *8 h, whereby the kits were
`obtained. The kits were stored at -4 °C temperature.
`
`Production and radiochemical processing of 177LuCl3
`
`Lu-177, which is regularly produced in our laboratory for the
`commercial deployment to various nuclear medicine centers
`[23], was used for the preparation of patient doses using the
`kits developed in-house. Typically, 200 lg of isotopically
`enriched Lu2O3 target (82 % in 176Lu) was irradiated at a
`thermal neutron flux of *9 9 1013 n cm-2 s-1 for a period
`of 21 days in our Institute’s reactor. The irradiated target was
`dissolved in 0.01 M supra-pure HCl by gentle warming. The
`resulting solution was evaporated to near dryness and
`reconstituted with supra-pure water. The evaporation and
`volume reconstitution steps were repeated two to three times
`in order to obtain 177LuCl3 in the pH range of 3–4. The
`radioactive solution was allowed to attain room temperature
`Ò
`(0.22 l)
`and subsequently passed through the Millipore
`filter paper in order to obtain 177LuCl3 in the sterile condi-
`tion. 177LuCl3, thus obtained, was directly used for the
`preparation of therapeutic doses of 177Lu-DOTA-TATE and
`177Lu-DOTA-NOC.
`The total 177Lu radioactivity produced and its radionu-
`clidic purity were determined following the procedure
`mentioned in the literature [24].
`
`Preparation of therapeutic dose of 177Lu-DOTA-TATE
`and 177Lu-DOTA-NOC using the freeze-dried kits
`
`Therapeutic dose of 177Lu-DOTA-TATE or 177Lu-DOTA-
`NOC was prepared by adding the required volume of
`177LuCl3 [200–400 lL, up to 200 mCi (7.4 GBq)] with the
`kit material dissolved in water for injection and subse-
`quently incubating the reaction mixture at 85–90 °C for a
`period of 45 min. Dissolution of the kit material was car-
`ried out using water such that the total volume of the
`preparation, after the addition of 177LuCl3, becomes 2 mL.
`When the radiochemical preparation attained room tem-
`perature, an aliquot was withdrawn and used to ascertain
`the radiochemical purity of the preparation using the
`quality control procedures mentioned below.
`
`Quality control studies
`
`The radiochemical purity of the complex was determined
`by PC and radio-HPLC. In PC, a small drop of the reaction
`mixture was spotted at 1.5 cm from one end of the chro-
`matography paper
`strip (10 9 1 cm). The strip was
`developed using 50 % aqueous acetonitrile (1:1, v/v) as the
`
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`solvent, dried, cut into 1 cm segments and the activity
`associated with each segment was recorded using NaI(Tl)
`detector.
`HPLC was carried out using a dual pump HPLC unit
`with a C-18 reversed phase HiQ-Sil (5 lM, 25 cm 9
`0.46 cm) column. Water (A) and acetonitrile (B) mixtures
`with 0.1 % trifluoroacetic acid were used as the mobile
`phase and the following gradient elution technique was
`adopted for the separation (0–4 min 95 % A, 4–15 min
`95 % A to 5 % A, 15–20 min 5 % A, 20–25 min 5 % A to
`95 % A, 25–30 min 95 % A). Flow rate was maintained at
`1 mL/min.
`
`Radiochemical studies
`
`In order to optimize the amount of DOTA coupled peptides
`required to be used in the kits, so that the radio-peptides
`could be prepared with adequately high radiochemical
`purity; attempt was made to prepare kits with different
`metal: DOTA-peptide molar ratios. While calculating the
`metal: DOTA-peptide molar ratios, it was considered that
`the kits will be used only when 177Lu will have a specific
`activity of C20 mCi/lg (740 MBq/lg). Freeze-dried kits,
`thus prepared with different amount DOTA coupled pep-
`tide were labeled with 177Lu and the corresponding radio-
`chemical purity of the preparation was determined by the
`quality control methods described above. All the radiola-
`beling experiments were carried out by incubating the
`ingredients at
`the reported optimized conditions i.e. at
`85–90 °C for a period of 45 min to 1 h [17, 19, 20]. The
`final pH of the reconstituted kit vial was automatically
`adjusted to the desired value i.e. *5, prior to incubation.
`
`Stability of 177Lu-DOTA-TATE and 177Lu-DOTA-
`NOC prepared using the freeze-dried kits
`
`The stability of 177Lu-DOTA-TATE and 177Lu-DOTA-
`NOC, prepared using the corresponding kits, was checked
`by storing the preparations at
`room temperature and
`determining the radiochemical purities of the preparations
`at different time intervals following the quality control
`procedures mentioned above.
`
`Biodistribution studies
`
`animals were used. The animals were sacrificed by over-
`dose of CO2 at 3 h, 1 d, 2 d and 7 d post-administration.
`Subsequent to sacrifice, the organs were excised, washed
`with saline, dried, weighed in a weighing balance and
`radioactivity associated with each organ was measured
`using a flat-type NaI(Tl) counter. Blood was collected
`immediately after sacrifice through cardiac puncture and
`counted in the same counter for determining the associated
`blood activity. The percentage of injected activity (%IA)
`accumulated in various organs/tissue was calculated from
`the above data. Total activity accumulated in the blood,
`muscle and bone was determined by considering the blood,
`muscle and bone weight to be 7, 40 and 10 % of the total
`body weight, respectively [25, 26]. The activity excreted
`was indirectly determined from the difference between
`total injected activity (IA) and %IA accounted for all the
`organs.
`
`Clinical studies
`
`Therapeutic doses of 177Lu-DOTA-TATE and 177Lu-
`DOTA-NOC (up to 200 mCi, 7.4 GBq) were prepared
`following the protocol described above and administered to
`the patients, suffering from cancers of neuroendocrine
`origin. About 1,000 mL of mixed amino acid solution
`(aminoven 10 %, composition:
`isoleucine 5 g,
`leucine
`7.4 g, lysine acetate 9.31 g, methionine 4.3 g, phenylala-
`nine 5.1 g; threonine 4.4 g, tryptophane 2 g; valine 6.2 g,
`arginine 12 g, histidine 3 g, alanine 14 g, glycine 11 g,
`proline 11.2 g, serine 6.5 g, tyrosine 0.4 g, taurine 1 g) was
`infused to each patient 4 h prior to administration of radio-
`peptides and continued for another 24 h after administra-
`tion to reduce the uptake in the kidneys. Post-therapy
`whole-body scans (1024 9 256 matrix size, scan speed
`15 cm/min) and SPECT-CT acquisition of the abdomen
`(128 9 128 matrix size, 20 s/projection, 16 projections)
`were performed using a dual-head gamma camera with
`high-energy-general-purpose collimators using the energy
`window centered on 113 and 208 keV photo-peaks of
`177Lu with a window width of ±20 %.
`
`Results and discussion
`
`Biological behaviors of 177Lu-DOTA-TATE and 177Lu-
`DOTA-NOC, prepared using the corresponding freeze-
`dried kits, were studied by carrying out biodistribution
`studies in normal Wistar rats each weighing 225–250 g.
`The radiochemical preparations were diluted with normal
`saline prior to administration in animals and each animal
`was injected with *3.7 MBq (100 lL, 100 lCi) of the
`preparation through the tail vein. For each time point, five
`
`Formulation of freeze-dried DOTA-TATE and DOTA-
`NOC kits
`
`Radiochemical studies carried out by labeling the kits
`having different molar ratios of DOTA coupled peptides
`and Lu showed that these two ingredients should be taken
`in at least 2:1 molar ratio in order to obtain 177Lu labeled
`peptides with [98 % radiochemical purity. This indicates
`that minimum 163 lg of DOTA-TATE or 165 lg of
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`1393
`
`0
`
`6
`5
`4
`3
`2
`1
`Distance from the point of spotting (cm)
`(a)
`
`7
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`% Complexation
`
`% Complexation
`
`0
`
`6
`5
`4
`3
`2
`1
`Distance from the point of spotting (cm)
`(b)
`
`7
`
`Fig. 1 PC profile of a 177Lu-DOTA-TATE and b 177Lu-DOTA-
`NOC, prepared using the corresponding freeze-dried kits
`
`activity C20 mCi/lg (740 MBq/lg). Typical HPLC pro-
`files of 177Lu-DOTA-TATE and 177Lu-DOTA-NOC, pre-
`pared using the cold kits, are shown in Fig. 2a, b,
`respectively.
`
`Stability of 177Lu-DOTA-TATE and 177Lu-DOTA-
`NOC prepared using the freeze-dried kits
`
`The stability of 177Lu-labeled peptides, prepared using the
`corresponding freeze-dried kits, was studied by storing the
`preparations at room temperature and determining the
`radiochemical purity of the preparations at different time
`intervals using the standard quality control
`techniques
`mentioned above. It was observed that both the complexes
`maintained their radiochemical purity of[99 % till 3 days
`post-preparation, up to which the study was continued.
`
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`DOTA-NOC has to be used in the kit in order to obtain
`177Lu labeled peptides with adequately high radiochemical
`purity when labeled with 177Lu having a minimum specific
`activity of 20 mCi/lg (740 MBq/lg). Therefore, each kit
`was formulated using 200 lg of DOTA-coupled peptide.
`Each kit vial actually comprises a lyophilized mixture of
`either 200 lg of DOTA-TATE or DOTA-NOC along with
`80 mg of gentisic acid and 13.9 mg of ammonium acetate.
`The kit vials were stored at -4 °C immediately after
`lyophilization. The vials were allowed to attain room
`temperature before the preparation of therapeutic doses of
`177Lu-DOTA-TATE or
`177Lu-DOTA-NOC for human
`administration.
`
`Production of 177Lu
`
`The kits were evaluated using the 177Lu obtained from dif-
`ferent batches having specific activities in the range of
`24.3 mCi/lg (899.1 MBq/lg) to 32.1 mCi/lg (1,187.7 MBq/
`lg). Lu-177m was found to be the only other radionuclide
`present in the processed 177Lu. After three weeks of irradiation
`at a thermal neutron flux of *9 9 1013 n cm-2 s-1, it was
`found that only 0.15 lCi (5.55 kBq) of 177mLu was present in
`per mCi (37 MBq) of 177Lu at the end-of-bombardment
`(EOB) [24]. This indicates that 177Lu was produced with
`[99.98 % radionuclidic purity at EOB. The radioactive
`concentration of the processed 177Lu was maintained between
`500 mCi/mL (18.5 GBq/mL) and 1 Ci/mL (37 GBq/mL)
`while the pH was between 3 and 4.
`
`Quality control studies
`
`In PC, carried out using 50 % aqueous acetonitrile (1:1,
`v/v) as the eluting solvent, it was observed that the activity
`corresponding to 177Lu-DOTA-TATE or 177Lu-DOTA-
`NOC moved towards the solvent front (Rf = 0.7 - 1),
`while uncomplexed 177Lu remained at the point of spotting
`(Rf = 0 - 0.1) under identical conditions. However, there
`exists a small difference between the Rf values exhibited by
`these two agents. While 177Lu-DOTA-NOC moved almost
`up to the solvent front (Rf = 0.8 - 1), 177Lu-DOTA-
`TATE exhibited Rf value in the range of 0.7 - 0.9. The
`typical PC patterns of 177Lu-DOTA-TATE and 177Lu-
`DOTA-NOC are shown in Fig. 1a, b, respectively. Similar
`difference was also observed in the retention time of 177Lu-
`DOTA-TATE and 177Lu-DOTA-NOC in radio-HPLC
`studies. It was observed that 177Lu-DOTA-TATE and
`177Lu-DOTA-NOC exhibit retention times of *18 and
`*20 min, respectively, while uncomplexed 177Lu was
`eluted from the column at *4 min. Radiochemical purity
`of 177Lu-labeled peptides was determined by employing
`the above two techniques and was found to be[99 % when
`radiolabeling was done by using 177Lu having specific
`
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`
`Fig. 2 HPLC profile of a 177Lu-DOTA-TATE and b 177Lu-DOTA-NOC, prepared using the corresponding freeze-dried kits
`
`Fig. 3 Stability of a 177Lu-DOTA-TATE and b 177Lu-DOTA-NOC, prepared using the corresponding freeze-dried kits, 3 days after preparation
`
`HPLC profiles showing the radiochemical purity of 177Lu-
`DOTA-TATE and 177Lu-DOTA-NOC after 3 days of
`storage at room temperature are shown in Fig. 3a, b,
`respectively.
`
`Biodistribution studies
`
`The comparative pharmacokinetic behavior of 177Lu-
`DOTA-TATE and 177Lu-DOTA-NOC was studied by
`
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`Table 1 Biodistribution pattern of 177Lu-DOTA-TATE, prepared using the freeze-dried DOTA-TATE kit, in normal Wistar rats
`
`Organ/tissue
`
`Injected activity per organ/tissue
`
`3 h
`
`1 d
`
`2 d
`
`7 d
`
`Blood
`
`Liver
`
`GIT
`
`Kidneys
`
`Stomach
`
`Heart
`
`Lungs
`
`Skeleton
`
`Muscles
`
`Spleen
`Excretiona
`
`0.57 ± 0.09
`
`0.80 ± 0.12
`
`3.92 ± 0.95
`
`4.46 ± 0.95
`
`0.24 ± 0.03
`
`0.02 ± 0.00
`
`0.11 ± 0.02
`
`2.18 ± 0.30
`
`1.71 ± 0.55
`
`0.03 ± 0.00
`
`0.00 ± 0.00
`
`0.54 ± 0.08
`
`4.21 ± 1.09
`
`4.98 ± 0.88
`
`0.40 ± 0.12
`
`0.00 ± 0.00
`
`0.03 ± 0.00
`
`0.05 ± 0.02
`
`0.62 ± 0.00
`
`0.02 ± 0.01
`
`0.00 ± 0.00
`
`0.24 ± 0.01
`
`0.50 ± 0.15
`
`1.43 ± 0.16
`
`0.04 ± 0.03
`
`0.00 ± 0.00
`
`0.00 ± 0.00
`
`0.00 ± 0.00
`
`0.00 ± 0.00
`
`0.04 ± 0.01
`
`0.00 ± 0.00
`
`0.26 ± 0.03
`
`0.15 ± 0.04
`
`0.20 ± 0.00
`
`0.02 ± 0.01
`
`0.00 ± 0.00
`
`0.00 ± 0.00
`
`0.00 ± 0.00
`
`0.00 ± 0.00
`
`0.07 ± 0.06
`
`86.66 ± 1.55
`
`89.21 ± 2.16
`
`97.75 ± 0.79
`
`98.11 ± 0.31
`
`Figures in the parentheses represent standard deviations. At every time point, three animals have been used
`a Excretion has been calculated by subtracting the activity accounted in all the organs from the total activity injected
`
`Table 2 Biodistribution pattern of 177Lu-DOTA-NOC, prepared using the freeze-dried DOTA-NOC kit, in normal Wistar rats
`
`Organ/tissue
`
`Injected activity per organ/tissue
`
`Blood
`
`Liver
`
`GIT
`
`Kidneys
`
`Stomach
`
`Heart
`
`Lungs
`
`Skeleton
`
`Muscles
`
`Spleen
`Excretiona
`
`3 h
`
`1.25 ± 0.30
`
`10.68 ± 1.28
`
`20.29 ± 0.76
`
`2.46 ± 0.48
`
`2.77 ± 0.71
`
`0.05 ± 0.01
`
`0.17 ± 0.03
`
`0.59 ± 0.28
`
`1.24 ± 0.02
`
`1.57 ± 0.27
`
`1 d
`
`0.26 ± 0.16
`
`8.62 ± 2.73
`
`14.04 ± 4.47
`
`2.91 ± 0.52
`
`1.40 ± 0.29
`
`0.01 ± 0.01
`
`0.19 ± 0.13
`
`0.96 ± 0.25
`
`0.75 ± 0.32
`
`1.00 ± 0.47
`
`2 d
`
`0.11 ± 0.02
`
`10.09 ± 0.78
`
`8.87 ± 2.40
`
`2.30 ± 0.20
`
`1.87 ± 0.57
`
`0.01 ± 0.01
`
`0.08 ± 0.02
`
`0.00 ± 0.00
`
`1.08 ± 0.51
`
`1.03 ± 0.48
`
`7 d
`
`0.00 ± 0.00
`
`3.16 ± 0.33
`
`1.94 ± 0.29
`
`2.31 ± 0.34
`
`0.09 ± 0.04
`
`0.00 ± 0.00
`
`0.07 ± 0.02
`
`0.00 ± 0.00
`
`1.07 ± 0.55
`
`0.11 ± 0.02
`
`58.41 ± 1.16
`
`69.84 ± 7.00
`
`74.71 ± 2.55
`
`85.91 ± 0.44
`
`Figures in the parentheses represent standard deviations. At every time point, three animals have been used
`a Excretion has been calculated by subtracting the activity accounted in all the organs from the total activity injected
`
`carrying out biodistribution studies in normal Wistar rats.
`The results of the biodistribution studies of 177Lu-DOTA-
`TATE and 177Lu-DOTA-NOC are shown in Tables 1 and 2,
`respectively. The studies revealed that there exist significant
`differences in the retention and clearance pattern of IA
`between 177Lu-DOTA-TATE and 177Lu-DOTA-NOC. It
`was observed that the retention of activity in liver and gas-
`trointestinal track is significantly higher in case of 177Lu-
`DOTA-NOC compared to that of 177Lu-DOTA-TATE at all
`the time points studied. After 7 d post-administration,
`3.16 ± 0.33 and 1.94 ± 0.29 %IA were retained in the liver
`and gastrointestinal track, respectively in case of 177Lu-
`DOTA-NOC; while the corresponding retentions were only
`0.26 ± 0.03 and 0.15 ± 0.04 %IA for 177Lu-DOTA-TATE.
`
`The retention of activity in stomach and spleen were also
`observed to be higher in case of 177Lu-DOTA-NOC com-
`pared to that of 177Lu-DOTA-TATE. 177Lu-DOTA-TATE
`showed lesser retention and faster clearance of blood activity
`than that of 177Lu-DOTA-NOC. The activity from the blood
`was completely cleared within 1 d post-administration in
`case of 177Lu-DOTA-TATE while retention of activity,
`though insignificant (0.11 ± 0.02 %IA), was observed in
`case 177Lu-DOTA-NOC even after 2 d post-administration.
`Renal clearance of 177Lu-DOTA-TATE was also found to be
`much faster compared to that of 177Lu-DOTA-NOC in all
`the time points studied. The marginally higher kidney uptake
`exhibited by 177Lu-DOTA-TATE in the initial time points
`was probably due to the much higher renal clearance shown
`
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`1396
`
`J Radioanal Nucl Chem (2014) 299:1389–1398
`
`Fig. 4 Whole-body scintigraphic images of patients treated with
`177Lu-DOTA-TATE and b 200 mCi
`a 200 mCi
`(7.4 GBq) of
`(7.4 GBq) of 177Lu-DOTA-NOC, prepared using the corresponding
`freeze-dried kits and recorded 6 h post-administration
`
`by this agent compared to that of 177Lu-DOTA-NOC.
`However, activity was observed to be retained for the longer
`time period in the kidneys in the case of 177Lu-DOTA-NOC
`(2.31 ± 0.34 %IA at 7 d p.i.) as compared to that of 177Lu-
`DOTA-TATE (0.20 ± 0.00 %IA at 7 d p.i.).
`
`Clinical studies
`
`In order to evaluate the comparative biological distribution
`of the radio-peptides, prepared using the kits,
`limited
`number of metastatic neuroendocrine cancer patients were
`administered 200 mCi (7.4 GBq) of the preparations. Post-
`therapy whole-body scans at different time points were
`recorded using the gamma camera and compared with the
`pre-therapy images obtained in the same patients using
`99mTc-HYNIC-TOC. Figure 4a represents whole-body
`scan of a patient (male, 40 years), suffering from recurrent
`pancreatic neuroendocrine tumor with recurrent inoperable
`pancreatic mass, recorded 6 h post-administration of 177Lu-
`DOTA-TATE. On the other hand, Fig. 4b represents sim-
`ilar time point post-therapy scan recorded with 177Lu-
`DOTA-NOC in a patient (female, 51 years), suffering from
`hepatic metastasis in a known case of neuroendocrine
`tumors. In both the cases higher uptake of the radiotracer in
`the cancerous lesions or metastatic sites was observed
`compared to liver or background on qualitative analysis.
`
`123
`
`Fig. 5 SPECT, CT, fusion and MIP images of the liver, acquired
`15 h after administration of 200 mCi (7.4 GBq) of a 177Lu-DOTA-
`TATE and b 177Lu-DOTA-NOC, prepared using the corresponding
`freeze-dried kits
`
`Moreover, it was observed that the distribution of the tracer
`in the post-therapy images was comparable for both the
`agents with that obtained in the pre-therapy images recorded
`with 99mTc-HYNIC-TOC. However, qualitative comparison
`between the images clearly indicate higher retention of
`activity in the blood, gastrointestinal track and kidneys in
`case of 177Lu-DOTA-NOC compared to that of 177Lu-
`DOTA-TATE, thereby corroborating the results obtained in
`the biodistribution studies. Figure 5a, b represent SPECT,
`
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`J Radioanal Nucl Chem (2014) 299:1389–1398
`
`1397
`
`CT, Fusion and MIP (Maximum Intensity Projection) ima-
`ges of the abdomen of patients injected with 200 mCi
`(7.4 GBq) of 177Lu-DOTA-TATE and 177Lu-DOTA-NOC,
`respectively, acquired 15 h post-administration. In both the
`cases, CT shows multiple hepatic lesions; while SPECT and
`fusion images demonstrate intense localization of the radio-
`peptides in the lesions. MIP images also exhibit intense
`hepatic uptake in both the cases. However, while only renal
`and splenic accumulation of activity was observed in case of
`177Lu-DOTA-TATE, accumulation of activity in bowel,
`kidneys and background soft
`tissue was additionally
`observed in case of 177Lu-DOTA-NOC.
`
`Conclusions
`
`Methodologies for the preparation of freeze-dried DOTA-
`NOC and DOTA-TATE kits, suitable for the formulation of
`therapeutic doses of 177Lu-DOTA-NOC and 177Lu-DOTA-
`TATE, respectively, have been developed. The cold kits were
`successfully utilized for the preparation of up to 200 mCi
`177Lu-labeled radio-peptides
`(7.4 GBq) of
`for human
`administration in a single and convenient step at the hospital
`radiopharmacy. Comparative biological evaluation of the two
`agents carried out in normal Wistar rats exhibited higher
`retention of activity in liver, gastrointestinal track and spleen;
`longer blood retention and slower renal clearance for 177Lu-
`DOTA-NOC compared to 177Lu-DOTA-TATE. Although
`both the radio-peptides exhibited accumulation in the can-
`cerous lesions, qualitative analyses of the scans showed higher
`retention and slower clearance of activity in case of 177Lu-
`DOTA-NOC compared to that of 177Lu-DOTA-TATE. Fur-
`ther studies are warranted to establish the use of 177Lu-DOTA-
`NOC in patients for treating neuroendocrine tumors.
`
`Acknowledgments The authors (T. Das, M. Bhadwal and S.
`Banerjee) are thankful
`to Dr. Gursharan Singh, Head,
`Isotope
`Applications and Radiopharmaceuticals Division (IA & RPhD) and
`Associate Director, Radiochemistry and Isotope Group, Bhabha Atomic
`Research Centre (BARC) for his support and interest. The authors are
`grateful
`to Dr. M.R.A. Pillai, former Head, Radiopharmaceuticals
`Division (presently known as IA & RPhD) BARC for the critical
`evaluation of the manuscript. The authors thankfully acknowledge the
`help received from the staff members of the animal house facility of
`BARC during the course of animal studies. The help rendered by the
`staff members of Kovai Medical Centre and Hospital while carrying out
`the human studi