ARTICLE IN PRESS
`
`Applied Radiation and Isotopes 65 (2007) 301–308
`
`www.elsevier.com/locate/apradiso
`
`On the preparation of a therapeutic dose of 177Lu-labeled
`DOTA–TATE using indigenously produced 177Lu in medium
`flux reactor
`
`Tapas Das, Sudipta Chakraborty, Sharmila Banerjee
`
`
`
`, Meera Venkatesh
`
`Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai-400085, India
`
`Received 26 June 2006; received in revised form 1 September 2006; accepted 29 September 2006
`
`Abstract
`177Lu could be produced with a specific activity of 23,000 mCi/mg (850 GBq/mg) by neutron activation using enriched 176Lu (64.3%)
`target when irradiation was carried out at a thermal neutron flux of 1  1014 n/cm2/s for 21 d. 177Lu–DOTA–TATE could be prepared in
`high radiochemical yield (99%) and adequate stability using the 177Lu produced indigenously. The average level of radionuclidic
`impurity burden in 177Lu due to 177mLu was found to be 250 nCi of 177mLu/1 mCi of 177Lu (9.25 kBq/37 MBq) at the end of
`bombardment, which corresponds to 0.025% of the total activity produced. The maximum specific activity achievable via careful
`optimization of
`the irradiation parameters was found to be adequate for the preparation of a therapeutic dose of
`the
`radiopharmaceutical. The in-house preparation of this agent using 25 mg (17.41 nmole) of DOTA–TATE and indigenously produced
`177Lu (0.8 mg, 4.52 nmole), corresponding to peptide/Lu ratio of 3.85 yielded 98.7% complexation. Allowing possibility of decay due to
`transportation to users, it has been possible to demonstrate that at our end, a single patient dose of 150–200 mCi (5.55–7.40 GBq) can be
`prepared by using 250–333 mg of DOTA–TATE conjugate. This amount compares well with 177Lu–DOTA–TATE prepared for a typical
`peptide receptor radionuclide therapy (PRRT) procedure which makes use of 100 mg of the DOTA–TATE conjugate, which incorporates
`50 mCi (1.85 GBq) of 177Lu activity, thereby implying that in order to achieve a single patient dose of 150–200 mCi (5.55–7.40 GBq),
`300–400 mg of the conjugate needs to be used.
`r 2006 Elsevier Ltd. All rights reserved.
`
`Keywords: PRRT; 177Lu; DOTA–TATE; Somatostatin receptors
`
`1. Introduction
`
`Radiometallated peptides which exhibit high specificity
`for cognate receptors over-expressed on cancerous lesions,
`offer important potential as site-directed diagnostic and
`therapeutic radiopharmaceuticals (Boerman et al., 2000;
`Breeman et al., 2001; Britton, 1997; Eckelman and Gibson,
`1993). The highly specific biochemical and physiologic
`binding capabilities of the receptor-avid peptides can be
`exploited for using these agents as a vehicle to target the
`delivery of the radioactivity to the cells over-expressing
`similar type of receptors after radiolabeling the peptides
`with the radionuclide of interest. Such peptide analogs
`
`
`
`Corresponding author. Tel.: +91 22 2559 0616; fax: +91 22 2550 5345.
`E-mail address: sharmila@barc.gov.in (S. Banerjee).
`
`0969-8043/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
`doi:10.1016/j.apradiso.2006.09.011
`
`when labeled with therapeutic radionuclides are being
`actively investigated as agents for use in peptide receptor
`radionuclide therapy (PRRT) (Kwekkeboom et al., 2000,
`2003a, 2005; de Jong et al., 2005).
`Among the several classes of radiolabeled peptides,
`radiolabeled somatostatin analogs have been proved to be
`the most promising. Octreotide, a metabolically stable
`analog of native somatostatin (Pauwels et al., 1998), has
`been radiolabeled with 111In using a bifunctional chelating
`agent and the resultant radiolabeled peptide, 111In–DTPA-
`Octreotide (commonly known as OctreoScans) has been
`successfully employed in clinical diagnosis of somatostatin
`receptor positive tumors (Pauwels et al., 1998; Krenning
`et al. 1992, 1993). Efforts are being made to develop suitable
`therapeutic analogs having the potential to eradicate the
`cancerous lesions over-expressing somatostation receptors.
`
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`Recently started clinical trials with (177Lu–DOTA–Tyr3)-
`Octreotide (known as 177Lu–DOTA–TOC) and (177Lu–
`DOTA–Tyr3)-Octreotate (known as 177Lu–DOTA–TATE)
`have shown very encouraging results in terms of tumor
`regression in patients suffering from different types of
`neuroendocrine tumors, which are known to over-express
`somatostatin receptors (Kwekkeboom et al., 2003a, b,
`2005; Forrer et al. 2005). However,
`it is reported that
`[DOTA–Tyr3]-Octreotate has a ninefold higher affinity for
`the somatostatin receptor subtype 2 as compared with
`[DOTA–Tyr3]-Octreotide and therefore, the later agent is
`expected to be more potent for carrying out PRRT in patients
`suffering from neuroendocrine tumors (Kwekkeboom et al.,
`2003a; Reubi et al. 2000).
`177Lu is presently being considered as a potential
`radionuclide for use in in vivo therapy, because of its
`favorable decay characteristics. 177Lu decays with a half-
`life of 6.73 d by emission of b- particles with maximum
`energies of 497 keV (78.6%), 384 keV (9.1%), and 176 keV
`(12.2%) to stable 177Hf (Firestone, 1996). The emission of
`suitable energy g photons of 113 keV (6.4%) and 208 keV
`(11%) (Firestone, 1996) with relatively low abundances
`provides
`the opportunity to carry out
`simultaneous
`scintigraphic studies, which helps to monitor the proper
`in vivo localization of the injected radiopharmaceutical as
`well as to perform dosimetric evaluations. An important
`aspect for the countries with limited reactor facility is the
`177Lu which provides
`comparatively long half-life of
`logistic advantage towards facilitating supply to locations
`far away from the reactors (Das et al., 2002). Besides this,
`the high thermal neutron capture cross-section (s ¼ 2100b)
`of (176Lu(n,g)177Lu) reaction (Firestone, 1996) ensures that
`177Lu can be produced with sufficiently high specific
`activity using the moderate flux reactors. In fact, the
`cross-section is the highest encountered among all (n,g)
`produced radionuclides presently used for therapy. These
`favorable nuclear parameters ensure that there will be no
`constraints with respect to large-scale isotope production.
`In connection with our ongoing research on the
`development of novel agents for radiotherapy of tumors
`(Banerjee et al., 2004; Das et al., 2004), production of
`177Lu,
`identified as
`the ideal
`radioisotope is being
`extensively optimized with an aim to achieving the
`maximum specific activity via a cost-effective route.
`This is an essential requirement for designing agents for
`PRRT since the primary criterion is to deliver sufficient
`number of radionuclides to the receptors over-expressed on
`the targeted tumor site without saturating the target
`(Mausner and Srivastava, 1993; Volkert and Hoffman,
`1999; Breeman et al., 2003).
`177Lu can be produced by two different routes, namely,
`by irradiation of Lu target and by irradiation of Yb target
`followed by radiochemical separation of 177Lu from Yb
`isotopes. Though the latter method enables production of
`no-carrier-added (NCA) 177Lu, the radiochemical separa-
`tion of 177Lu activity from irradiated Yb target is a difficult
`task owing to the similarity in the chemistry of the two
`
`adjacent members of the lanthanide series. Presence of Yb
`in substantial amounts, attributable to its low cross-
`section, besides reducing the effective specific activity of
`the product, will also compete with 177Lu in the complexa-
`tion procedure unless effectively eliminated from the
`irradiated target. This is one of the major impediment in
`
`the ready production of NCA 177Lu via 176Yb(n,g,b
`)177Lu
`route. Moreover, use of enriched targets with low activation
`cross-section (s ¼ 2.4b for
`176Yb(n,g)177Yb)
`(Firestone,
`1996) is not economical for isotope production, as a
`significant part of the target remains unutilized. Though,
`176Yb recovery is theoretically feasible, the aforementioned
`practical problems associated with the cumbersome separa-
`tion still remains.
`177Lu have
`in the production of
`Our experiences
`provided an insight towards envisaging 177Lu–DOTA–
`TATE as a radiopharmaceutical, which can be indigen-
`ously produced and supplied to local users as an agent for
`PRRT. The primary aim in the designing of this agent
`attempts to maximize the specific activity of the resultant
`radiolabeled preparation. Towards this, 177Lu production
`is envisaged via (n,g) using an enriched Lu2O3 target and
`efforts are directed to obtain 177Lu with maximum possible
`specific activity utilizing the moderate flux reactor available
`in our country. However, careful optimization of the
`irradiation parameters is required for obtaining 177Lu in
`adequate specific activity for PRRT applications, particu-
`larly when production is envisaged at high flux positions
`owing to the high thermal neutron capture cross-section of
`176Lu which eventually leads to considerable target burn
`up. The present paper describes optimization of 177Lu
`production with sufficiently high specific activity using a
`moderate flux reactor and successful preparation of patient
`dose of 177Lu–DOTA–TATE using the 177Lu indigenously
`produced at our end.
`
`2. Materials and methods
`
`Lutetium oxide (64.3% enriched in 176Lu, spectroscopic
`grade, 499.99% pure) was obtained from Isoflex, Russia.
`DOTA–TATE was obtained from PiChem, Finland,
`through International Atomic Energy Agency (IAEA) as
`a part of a coordinated research project
`(CRP). All
`chemicals and solvents used in the experiments were of
`AR grade and supplied by reputed chemical manufac-
`turers. Radionuclidic purity of 177Lu was ascertained by
`high resolution g-ray spectrometry using an HPGe detector
`(EGG Ortec/Canberra detector) coupled to a 4K multi-
`channel analyzer
`(MCA)
`system after
`radiochemical
`processing. 152Eu reference source used for energy and
`efficiency calibration of the detector was obtained from
`Amersham Inc., USA. All other radioactivity measure-
`ments were carried out using a well
`type NaI (Tl)
`scintillation counter after adjusting the 150 keV baseline
`and keeping a window of 100 keV, thereby utilizing the
`208 keV g photon of 177Lu. Whatman 3 mm chromato-
`graphy paper (UK) was used for paper chromatography
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`303
`
`studies. The high performance liquid chromatography
`(HPLC) system used was obtained from JASCO (PU
`1580), Japan, equipped with a PU 1575 UV/VIS detector.
`A well-type NaI (Tl) scintillation detector was coupled to
`the system for radioactivity measurements in the eluate. All
`the solvents used for HPLC analyses were of HPLC grade
`and purchased from reputed local manufacturers, degassed
`and filtered prior to use.
`
`2.1. Production and radiochemical processing of 177Lu
`
`177Lu was produced by thermal neutron bombardment
`on isotopcially enriched (64.3% in 176Lu) Lu2O3 target. A
`stock solution of enriched target was prepared by dissol-
`ving enriched Lu2O3 powder in 0.1 M HCl (1 mg/mL
`concentration). A known aliquot of this solution was taken
`in a quartz ampoule and carefully evaporated to dryness.
`The ampoule was subsequently flame sealed and irradiated
`after placing inside an aluminum can. Irradiations were
`carried out at different available flux positions (1.4 
`1013–1.0  1014 n/cm2/s) for different durations (7–21 d) in
`order to optimize the irradiation conditions towards
`achieving maximum specific activity of 177Lu. The irra-
`diated target was dissolved in 1 M HCl by gentle warming
`inside a lead-shielded plant. The resultant solution was
`evaporated to near dryness and reconstituted in de-ionized
`water. The pH of the 177LuCl3 solution thereby obtained
`was adjusted to 4 prior to complexation studies. A known
`aliquot was drawn for assay of radioactivity and determi-
`nation of radionuclidic purity.
`Radioactivity assay was carried out by measuring the
`ionization current obtained when an aliquot of the batch
`was placed inside a pre-calibrated well-type ion-chamber.
`Radionuclidic purity was determined by recording g-ray
`spectra of
`the appropriately diluted solution of
`the
`irradiated target using an HPGe detector connected to a
`4 K MCA system. Energy as well as efficiency calibration of
`the detector were carried out using a 152Eu reference source
`prior to the recording of g-ray spectra. Several spectra were
`recorded for each batch at regular time intervals. Samples
`measured initially for the assay of 177Lu were preserved for
`complete decay of 177Lu (8–10 T1/2 of 177Lu, i.e. for a period
`of 50–70 d) and re-assayed to determine the activity of long-
`lived 177mLu (T1/2 ¼ 160.5 d). Appropriately diluted sample
`
`solutions were counted for 1 h.
`
`2.2. Preparation of 177Lu–DOTA–TATE complex
`
`177Lu-labeled DOTA–TATE
`the preparation of
`For
`complex, a stock solution of the DOTA–TATE was prepared
`by dissolving DOTA–TATE conjugate in HPLC grade water
`with a concentration of 1 mg/mL. To a 25 mL aliquot of this
`stock solution, 25 mL of 177LuCl3 solution was added and the
`volume was made upto 200 mL using the 0.1 M ammonium
`acetate buffer of pH 5. The reaction mixture was incubated
`at 80 1C for 1 h after adjusting the pH to 4.5–5. Various
`parameters, such as, ligand concentration, incubation time,
`
`and temperature were varied extensively in order to arrive at
`the protocol for maximum complexation.
`
`2.3. Quality control techniques
`
`The characterization of the 177Lu-labeled DOTA–TATE
`conjugate as well as the determination of the complexation
`yield was carried out by paper chromatography (PC) as
`well as by HPLC techniques.
`
`2.3.1. Paper chromatography (PC)
`5 mL portions of the test solutions were applied at 1.5 cm
`from the lower end of the chromatography paper strips.
`The strips were developed in 50% aqueous acetonitrile,
`dried, cut into segments of 1 cm each and the radioactivity
`associated with each segment was measured in a NaI (Tl)
`detector.
`
`2.3.2. High performance liquid chromatography (HPLC)
`HPLC of the 177Lu-labeled conjugate was carried out
`using a dual pump HPLC unit with a C-18 reversed phase
`HiQ-Sil (5 mm, 25  0.46 cm) column. The elution was
`monitored both by detecting UV signals at 270 nm as well
`as by radioactivity signal using NaI (Tl) detector. 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.
`
`2.4. Maximization of specific activity of
`177Lu–DOTA–TATE complex
`
`In order to obtain 177Lu-labeled DOTA–TATE con-
`jugate in highest possible specific activity, experiments were
`carried out to achieve maximum complexation yield of
`177Lu-DOTA–TATE at
`the minimum possible [L]/[M]
`ratio. For this, 25 mg of the peptide (already optimized
`ligand amount) was allowed to react with different
`amounts of Lu ranging from 0.2 to 1.6 mg under the same
`reaction conditions described earlier.
`
`2.5. Stability of 177Lu–DOTA–TATE
`
`The in vitro stability of the 177Lu-labeled conjugate was
`ascertained by storing the radiolabeled product at room
`temperature and determining the radiochemical purity at
`different time intervals post-preparation employing the
`standard quality control techniques described earlier.
`
`3. Results and discussions
`
`3.1. Production of 177Lu
`
`177Lu was produced by thermal neutron bombardment
`on isotopically enriched (64.3% in 176Lu) Lu2O3 target in
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`Dhruva reactor at our Institute. The radionuclidic purity of
`177Lu produced was determined by analyzing the g-ray
`spectrum and found to be 99.975%. A typical g-ray
`spectrum recorded after the radiochemical processing of
`the irradiated target exhibited major g photopeaks at 72,
`113, 208, 250, and 321 keV. All of these photopeaks
`correspond to that of 177Lu (Firestone, 1996). This was
`further confirmed from the decay as followed by monitor-
`ing peak area cps values at those peaks according to the
`half-life of 177Lu. It is well documented in the literature
`that there is a possibility of formation of 177mLu (T1/2
`¼ 160.5 d) on thermal neutron bombardment of Lu target
`(Neves et al., 2002; Pillai et al., 2003; Nir-El, 2004).
`However, g-ray spectrum of the irradiated Lu target after
`chemical processing did not show any significant peak
`corresponding to the photopeaks of 177mLu (128, 153, 228,
`378, 414, 418 keV) (Firestone, 1996). This can be explained
`from the fact that the radioactivity due to 177mLu produced
`will be insignificant compared to that of 177Lu, owing to its
`long half-life
`and comparatively
`low cross-section
`(s ¼ 7 barns) (Firestone, 1996) for its formation. The trace
`level of 177mLu produced was determined by recording the
`g-ray spectrum of a sample aliquot, initially having high
`radioactive concentration, after complete decay of 177Lu
`activity (8–10 T1/2 of 177Lu, i.e. for a period of 50–70 d).
`The average level of radionuclidic impurity burden in 177Lu
`due to 177mLu determined by this technique was found to
`be 250 nCi of 177mLu/1 mCi of 177Lu (9.25 kBq/37 MBq) at
`EOB, which corresponds to 0.025% of the total activity
`produced.
`In order to produce 177Lu with maximum possible
`specific activity using the moderate flux reactor available at
`our end, Lu2O3 target was irradiated at different available
`flux positions for different periods of time. Four different
`flux positions with thermal neutron flux ranging between
`1.4  1013 and 1.0  1014 n/cm2/s were available for irradia-
`tion. Since the normal irradiation schedule of this multi-
`facility reactor allowed irradiation of the target only in
`
`multiples of 7 d, irradiations were carried out for 7, 14, and
`the 177Lu obtained on
`21 d. The specific activity of
`irradiation of enriched target for varied time durations
`and at different
`thermal neutron flux positions are
`tabulated in Table 1. A maximum specific activity of
`23000 mCi/mg (850 GBq/mg) was achieved when irradia-
`tion was carried out at a thermal neutron flux of 1 1014 n/
`cm2/s for 21 d, which corresponds to 21% of
`the
`maximum achievable specific activity. It is evident from
`Table 1 that the specific activities of 177Lu obtained were
`significantly higher compared to the theoretically calcu-
`lated values accounting for only thermal neutron capture.
`A possible reason for practically obtaining an activity
`higher
`than theoretically calculated values could be
`attributed to the contribution from epithermal neutrons
`(resonance integral ¼ 1087b), which is not accounted for in
`theoretical calculations (Pillai et al., 2003; Nir-El, 2004;
`Knapp et al., 1995). However, the contribution from
`epithermal neutron alone could not provide a satisfactory
`explanation for obtaining 2.5–2.8 times higher specific
`activity of 177Lu than that of
`theoretically calculated
`values.
`Apart from the use of enriched target and choosing
`the highest available neutron flux position in a reactor,
`the other factor, which can be optimized for achieving the
`maximum possible specific activity of a radioisotope
`produced by neutron activation,
`is
`the duration of
`irradiation. In most cases of radioisotope production using
`(n,g) route, irradiation of the target 4–6 times of the half-
`life of the radioisotope being produced results in maximum
`specific activity. However, the same does not hold good in
`case of production of 177Lu by neutron capture, particu-
`larly, when the production is being carried out at a
`comparatively higher neutron flux position, owing to the
`high thermal neutron capture cross section of 177Lu. In this
`case, a careful optimization of the time of irradiation needs
`to be considered in order to obtain the highest specific
`activity (Pillai et al., 2003). In high flux positions of a
`
`Table 1
`Specific activity of 177Lu obtained at different thermal neutron flux for irradiation of different durations
`
`Neutron flux (n/cm2/s)
`
`Irradiation time (d)
`
`Activity obtained
`(mCi/mg) (GBq/mg)
`
`Theoretical activity
`(mCi/mg) (GBq/mg)
`
`Experimental/theoretical
`
`1.4 1013
`
`3.0 1013
`
`3.0 1013
`
`6.75 1013
`
`6.75 1013
`
`1.0 1014
`
`7
`
`7
`
`14
`
`7
`
`14
`
`21
`
`23227102
`85.973.8
`
`46357269
`171.579.9
`
`74607253
`276.079.4
`
`110007782
`407.0728.9
`
`1775071476
`656.8754.6
`
`231857658
`857.8724.3
`
`846
`31.3
`
`1814
`67.1
`
`2696
`99.8
`
`4081
`151.0
`
`6406
`237.0
`
`8622
`319.0
`
`2.74
`
`2.56
`
`2.77
`
`2.70
`
`2.77
`
`2.69
`
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`
`3.2. Characterization of 177Lu–DOTA–TATE
`
`In PC using 50% aqueous acetonitrile, the activity
`corresponding to 177Lu–DOTA–TATE complex moved
`towards the solvent front with Rf ¼ 0.8–0.9, while un-
`complexed 177Lu remained at the point of spotting (Rf ¼ 0)
`under identical conditions. The PC patterns of 177LuCl3
`and 177Lu–DOTA–TATE complex are shown in Fig. 2.
`The 177Lu-labeled DOTA–TATE complex was also
`characterized by HPLC studies. Fig. 3(a) shows the typical
`HPLC pattern of 177Lu-labeled DOTA–TATE conjugate
`prepared under optimized conditions. Fig. 3(b) shows the
`HPLC pattern of 177LuCl3 under identical conditions. The
`actual extent of complexation achieved was also deter-
`mined from the HPLC studies.
`
`3.3. Optimization of complexation yield of
`177Lu–DOTA–TATE
`
`Various parameters such as, conjugate concentration,
`pH of the reaction mixture, reaction time and temperature
`were varied extensively in order to achieve maximum
`complexation yield. Keeping the reaction volume as
`200 mL, the amount of DOTA–TATE was varied from 5
`to 100 mg in order to determine the optimum ligand
`concentration required for obtaining maximum complexa-
`tion. It was observed that a minimum of 25 mg DOTA–
`TATE was required to obtain a complexation yield of
`99%. The reaction was carried out by incubating the
`reaction mixture at three different temperatures (room
`temperature, 50 and 80 1C) for different time periods
`(5 min, 15 min, 30 min, 1 h, and 2 h) in order to determine
`optimum reaction time and temperature. It was observed
`that, maximum complexation was achieved when the
`reaction mixture was incubated at 80 1C for a period of
`1 h. It
`is well documented in the literature that
`the
`
`177Lu-DOTA-TATE
`
`177LuCl3
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`% Radioactivity
`
`0
`
`1
`
`2
`3
`4
`5
`Migration from point of spotting (cm)
`
`6
`
`7
`
`Fig. 2. Paper chromatography patterns of 177LuCl3 and 177Lu–DOTA–
`TATE.
`
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`
`reactor, the target burn up will be considerably higher
`owing to the high thermal neutron capture cross section
`of 176Lu and hence, the usual assumption that the number
`of target atoms remains constant during the period of
`irradiation will not be valid in this case. Considering the
`fact that the number of target atoms is not fixed but a
`function of time, the commonly used differential equation
`used for the calculation of the activity produced i.e.
`
`dN 2=dt ¼ N 1sf N 2l
`
`(1)
`
`can be modified as
`
`sftsf N 2l,
`dN 2=dt ¼ N 0e
`(2)
`where N0 is the number of 176Lu atoms used as target (at
`t ¼ 0), N1 the number of 176Lu atoms at any time t, N2 the
`number of 177Lu atoms at any time t, l the decay constant
`of 177Lu, s the thermal neutron capture cross section of
`176Lu, f the thermal neutron flux of the reactor and t the
`time of
`irradiation. Solution of
`the above mentioned
`modified differential equation leads
`to the following
`mathematical expression, which enables calculation of the
`177Lu activity (A) produced at EOB, taking into account
`that the number of the target atoms is also a function of
`time of irradiation.
`½e
`sft e
`A ¼ N 0lsf
`l sf
`Fig. 1 shows the variation of the theoretically calculated
`specific activity of 177Lu obtained at EOB with irradiation
`time at 1 1014 n/cm2/s thermal neutron flux. It is evident
`that the activity of 177Lu produced will be maximum after
`21 d irradiation, beyond which, the activity will decrease
`owing to the high target burn up. Therefore, in order to
`obtain maximum specific activity of 177Lu using the
`maximum available flux position in our reactor, the time
`of irradiation was fixed as 21 d.
`
`ltŠ
`
`(3)
`
`0
`
`7
`
`14
`Duration of irradiation (d)
`
`21
`
`28
`
`325
`
`300
`
`275
`
`250
`
`225
`
`200
`
`175
`
`150
`
`125
`
`100
`
`Specific activity of177Lu (GBq/mg)
`
`Fig. 1. Variation of theoretically calculated specific activity of 177Lu
`obtained at EOB with time of irradiation at thermal neutron flux of
`1  1014 n/cm2/s.
`
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`
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`
`is kept constant at this value and complexations were
`carried out using different amounts of Lu ranging from 0.2
`to 1.6 mg under the same reaction conditions described
`earlier. The complexation yield in each case was deter-
`mined by PC as well as by HPLC. The complexation yields
`obtained at different concentrations of Lu are given in
`Table 2. It is evident from Table 2, that a radiolabeling
`yield of 99% could be achieved using a ligand/metal ratio
`4. This implies that 0.8 mg of Lu can be incorporated in
`25 mg of the peptide without compromising the extent of
`complexation. Considering the maximum possible specific
`activity of 177Lu achievable at our end to be 23,000 mCi/mg
`(850 GBq/mg), 0.8 mg of Lu could provide 18.4 mCi
`(0.68 GBq) of activity. Therefore, a single patient dose of
`150–200 mCi (5.55–7.40 GBq) can be prepared by using
`200–275 mg of peptide. In order to carry out a comparative
`evaluation of the radiolabeled DOTA–TATE prepared by
`us, an analysis of a reported case (Kwekkeboom et al.,
`2003a, 2001) was carried out. It has been shown that
`177Lu–DOTA–TATE prepared for a typical PRRT pro-
`cedure uses 100 mg of
`the DOTA–TATE conjugate
`which incorporates 50 mCi (1.85 GBq) of 177Lu activity
`(Kwekkeboom et al., 2001). This implies that to achieve a
`single patient dose of 150–200 mCi
`(5.55–7.40 GBq),
`300–400 mg of the conjugate has been used. Considering
`the practical aspects of time required for Post-irradiation
`radiochemical processing, preparation and quality control
`of the radiopharmaceutical and subsequent transportation
`to the nuclear medicine facilities, 15 mCi (0.55 GBq) of
`177Lu activity in 25 mg of DOTA–TATE can be delivered to
`the nuclear medicine facilities. Therefore, the single patient
`dose of 150–200 mCi (5.55–7.40 GBq) can be prepared by
`using 250–333 mg of DOTA–TATE conjugate at our end,
`which is comparable or possibly less than the reported
`values.
`
`3.5. Stability of 177Lu–DOTA–TATE
`
`The stability of the 177Lu-labeled DOTA–TATE complex
`was studied by employing standard quality control techni-
`ques mentioned earlier. It was observed that the radi-
`olabeled conjugate retained its radiochemical purity after
`7 d of its preparation when stored at room temperature.
`
`Table 2
`Complexation yield of 177Lu–DOTA–TATE at different [L]/[M] ratio
`
`[DOTA–TATE]
`
`[Lu]
`
`[L]/[M]
`
`Complexation
`yield (%)
`
`25 mg
`(17.41 nmole)
`25 mg
`(17.41 nmole)
`25 mg
`(17.41 nmole)
`25 mg
`(17.41 nmole)
`
`0.2 mg
`(1.13 nmole)
`0.4 mg
`(2.26 nmole)
`0.8 mg
`(4.52 nmole)
`1.6 mg
`(9.04 nmole)
`
`15.41
`
`7.70
`
`3.85
`
`1.92
`
`99.2
`
`99.0
`
`98.7
`
`81.2
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`0
`
`0
`
`300
`
`1200
`900
`600
`Retention time (s)
`
`1500
`
`1800
`
`CPS
`
`(a)
`
`400
`
`300
`
`200
`
`CPS
`
`100
`
`0
`
`0
`
`300
`
`(b)
`
`1200
`900
`600
`Retention time (s)
`
`1500
`
`1800
`
`Fig. 3. HPLC patterns of (a) 177Lu–DOTA–TATE and (b) 177LuCl3.
`
`maximum complexation is obtained when 177Lu labeling of
`the biomolecules are carried out at pH5 (Breeman et al.,
`2003; Milenic et al., 2002; Stimmel and Kull, 1998). In the
`present case,
`the maximum complexation yield was
`achieved at pH 4.5–5.
`
`3.4. Maximization of the specific activity of
`177Lu–DOTA–TATE
`
`Bearing in mind the importance of preparing radiola-
`beled agents intended for targeted use in binding with
`receptors over-expressed on tumors, efforts were directed
`to prepare the 177Lu-DOTA–TATE with maximum possi-
`ble specific activity, using the 177Lu available at our end.
`Towards achieving this, the concentration of the peptide
`conjugate used for a typical labeling procedure needs to be
`minimum which,
`in turn, would aim at the minimum
`possible ligand/metal ratio, in order that unlabeled peptide
`which would otherwise bind also with the targeted
`receptors will be minimum. The ligand/metal ratio can be
`minimized by keeping the concentration of the peptide
`constant and gradually increasing the amount of radio-
`metal in the reaction mixture. Since 25 mg of the peptide
`was found to be the optimum amount required for
`obtaining 99% complexation, the ligand concentration
`
`Evergeen Ex. 1031
`6 of 8
`
`

`

`ARTICLE IN PRESS
`
`T. Das et al. / Applied Radiation and Isotopes 65 (2007) 301–308
`
`307
`
`The complex prepared using maximum possible amount of
`Lu (0.8 mg) also showed excellent stability till 7 d when
`stored at identical conditions.
`
`4. Conclusion
`
`Production of 177Lu by the (n,g) route from enriched
`176Lu target with a specific activity of 23000 mCi/mg
`(850 GBq/mg) was achievable when irradiation was carried
`out at a thermal neutron flux of 1  1014 n/cm2/s for 21 d.
`177Lu–DOTA–TATE could be prepared in high radio-
`chemical yield and adequate stability using the 177Lu
`produced indigenously. The maximum specific activity
`achievable via careful optimization of
`the irradiation
`parameters was found to be adequate for the preparation
`of a therapeutic dose of the radiopharmaceutical. The in-
`house preparation of
`this agent using indigenously
`produced 177Lu was found to be a more cost-effective
`route over the method involving use of 177Lu from
`commercial sources. Clinical studies with this agent will
`be carried out after obtaining clearances from relevant
`regulatory authorities.
`
`Acknowledgments
`
`The authors sincerely acknowledge the support provided
`by the International Atomic Energy Agency (IAEA) in the
`form of a coordinated research project (CRP). The authors
`express their sincere thanks to Dr. V. Venugopal, Director,
`Radiochemistry and Isotope Group for his valuable
`suggestions and constant encouragement through out the
`course of the work. The sincere help received from our
`colleagues, Dr. S.V. Thakare and Mr. K.C. Jagadeesan for
`carrying out
`irradiations of
`lutetium oxide target
`is
`gratefully acknowledged.
`
`References
`
`Banerjee, S., Das, T., Chakraborty, S., Samuel, G., Korde, A., Srivastava,
`S., Venkatesh, M., Pillai, M.R.A., 2004. 177Lu–DOTA–lanreotide: a
`novel tracer as a targeted agent for tumor therapy. Nucl. Med. Biol.
`31, 753–759.
`Boerman, O.C., Oyer, W.J.G., Corstens, F.H.M., 2000. Radio-labeled
`receptor-binding peptides: a new class of radiopharmaceuticals. Semin.
`Nucl. Med. 30, 195–208.
`Breeman, W.A.P., de Jong, M., Kwekkeboom, D.J., Valkema, R., Kooij,
`P.P., Visser, T.J., Krenning, E.P., 2001. Somatostatin receptor-
`mediated imaging and therapy: basic science, current knowledge,
`limitations and future perspectives. Eur. J. Nucl. Med. 28, 1421–1429.
`Breeman, W.A.P., de Jong, M., Visser, T.J., Erion, J.L., Krenning, E.P.,
`2003. Optimising conditions for radiolabelling of DOTA-peptides with
`90Y, 111In and 177Lu at high specific activities. Eur. J. Nucl. Med. Mol.
`Imag. 30, 917–920.
`Britton, K.E., 1997. Towards the goal of cancer specific imaging and
`therapy. Nucl. Med. Commun. 18, 992–1005.
`Das, T., Chakraborty, S., Unni, P.R., Banerjee, S., Samuel, G., Sarma,
`H.D., Venkatesh, M., Pillai, M.R.A., 2002. 177Lu labeled cyclic
`polyaminophosphonates as potential agents for bone pain palliation.
`Appl. Radiat. Isot. 57, 177–184.
`
`de
`
`Das, T., Chakraborty, S., Banerjee, S., Mukherjee, A., Samuel, G., Sarma,
`H.D., Nair, C.K.K., Kagiya, V.T., Venkatesh, M., 2004. Preparation
`and preliminary biological evaluation of a 177Lu labeled sanazole
`derivative for possible use in targeting tumor hypoxia. Bioorg. Med.
`Chem. 12, 6077–6084.
`Jong, M., Breeman, W.A.P., Valkema, R., Bernard, B.F.,
`Krenning, E.P., 2005. Combination radionuclide therapy using
`177Lu- and 90Y-labeled somatostatin analogues. J. Nucl. Med. 46,
`13S–17S.
`Eckelman, W.C., Gibson, R.E., 1993. The design of site-directed radio-
`pharmaceuticals for use in drug discovery. In: Burns, H.D., Gibson,
`R.E., Dannals, R., Siegl, P.
`(Eds.), Nuclear Imaging in Drug
`Discovery, Development, and Approval. Birkhauser, Boston.
`Firestone, R., 1996. In: Shirley, V.S. (Ed.), Table of Isotopes, eighth ed.
`Wiley, New York.
`Forrer, F., Uusijarvi, H., Storch, D., Maecke, H.R., Mueller-Brand, J.,
`2005. Treatment with 177Lu–DOTATOC of patients with relapse of
`neuroendocrine tumors after treatment with 90Y–DOTATOC. J. Nucl.
`Med. 46, 1310–1316.
`Knapp, F.F., Ambrose, K.R., Beets, A.L., Luo, H., McPherson, D.W.,
`Mirzadeh, S., 1995. Nuclear medicine program progress report for
`quarter ending September 30, 1995. Oak Ridge National Laboratory,
`pp. 6–16.
`Krenning, E.P., Bakker, W.H., Kooij, P.P., Breeman, W.A., Oei, H.Y., de
`Jong, M., Reubi, J.C., Visser, T.J., Bruns, C., Ikekubo, K., 1992.
`Somatosta