Applied Radiation and Isotopes 85 (2014) 28–33
`
`Contents lists available at ScienceDirect
`
`Applied Radiation and Isotopes
`
`journal homepage: www.elsevier.com/locate/apradiso
`
`Application of single-vial ready-for-use formulation
`of 111In- or 177Lu-labelled somatostatin analogs
`Erik de Blois, Ho Sze Chan, Rory de Zanger, Mark Konijnenberg, Wouter A.P. Breeman n
`
`Erasmus MC Rotterdam, Department of Nuclear Medicine, ‘s Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands
`
`H I G H L I G H T S
` Optimal quencher combination: ascorbic- and gentisic acid and ethanol.
` Used quencher concentrations had no effect on pharmacokinetics.
` Purging the reaction mixture with N2 after radiolabelling resulted in 10% higher RCP.
` Quencher mixture stabilize 111In- and 177Lu-labelled SS-analogs during 7 days.
` Enables to store and transport 111In- and 177Lu-labelled SS-analogs in a single-vial.
`
`a r t i c l e i n f o
`
`a b s t r a c t
`
`For the sake of safety it would be desirable to store and transport the ready-for-use liquid formulation
`(diagnostics and therapeutics) of radiolabelled peptides. The use of ethanol, in combination with a
`mixture of gentisic- and ascorbic acid, has superior effects on stabilizing radiolabelled somatostatin
`analogs. As a consequence, 111In- and 177Lu-labelled somatostatin analogs can be stored and transported
`in a single-vial ready-for-use liquid formulation up to 7 days after radiolabelling.
`& 2013 Elsevier Ltd. All rights reserved.
`
`Article history:
`Received 14 June 2013
`Received in revised form
`4 October 2013
`Accepted 26 October 2013
`Available online 10 December 2013
`
`Keywords:
`Somatostatin
`Quenchers
`Ethanol
`Ascorbic acid
`Gentisic acid
`Radiochemical purity
`
`1.
`
`Introduction
`
`Here we describe the presence of quenchers (de Blois et al.,
`2012) in a single-vial
`liquid pharmaceutical formulation of a
`radiolabelled peptide, in a quantity sufficient to prevent radiolysis
`of the formulation. Radiolabelled peptides are generally stored and
`transported in the form of multi-vial kit formulations. Usually the
`contents of these vials are lyophilized or frozen and should be
`brought into solution subsequently in a mutual reaction to
`produce the intended radiolabelled peptide. For the sake of safety
`it would be desirable to be able to store and transport the ready-
`for-use liquid formulation of the radiolabelled peptide. Then, a
`physician could administer the labelled peptide without a radio-
`chemical reaction, simply by diluting the contents of the vial in a
`radiopharmaceutical liquid that can be administered by injection
`or by infusion (Filice et al., 2012).
`
`n Corresponding author. Tel.: þ31 10 7035317; fax: þ31 10 7035997.
`E-mail address: w.a.p.breeman@erasmusmc.nl (W.A.P. Breeman).
`
`0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
`http://dx.doi.org/10.1016/j.apradiso.2013.10.023
`
`During labeling, storage and transport of the somatostatin
`analogs (further referred as SS-analogs) the peptide is exposed
`to radicals produced by i.e. 177Lu or 111In as radionuclide. Radiolysis
`of aqueous solutions produces reactive species (∙OH, H∙, e
`aq, H2O2)
`that may react with the peptide in the reaction (Garrison, 1987;
`Jay-Gerin and Ferradini, 2000; Jonah, 1995; Swiatla-Wojcik and
`Buxton, 2005). Here we describe how to preserve high radio-
`chemical purity – and thus to increase the storage and transport
`time of radiolabelled SS-analogs – with the use of quenchers. The
`literature reports successful addition of quenchers such as gentisic
`acid (Liu and Edwards, 2001), ascorbic acid (Chen et al., 2008; Liu
`et al., 2001, 2003), methionine (Breeman et al., 2008) and ethanol
`(Chen et al., 2008; Filice et al., 2012; Fukumura et al., 2004;
`Schuessler, 1975; Erion et al., 2008) to the reaction mixtures in
`various combinations and concentrations prior to radiolabelling to
`prevent radiolysis.
`In this study, stability and radiolysis of radiolabelled SS-analogs
`were monitored by HPLC. HPLC methods were optimized to
`distinguish between non-labelled and radiolabelled peptides vs.
`the radiolysed peptides. The effect of quenchers on the stability of
`
`Evergeen Ex. 1017
`1 of 6
`
`

`

`E. de Blois et al. / Applied Radiation and Isotopes 85 (2014) 28–33
`
`29
`
`radiolabelled SS-analogs, even under therapeutic conditions was
`optimized and monitored up to 7 days after radiolabelling.
`
`2. Methods and materials
`
`2.1. 111In/177Lu labelling of SS-analogs
`
`DOTA-TATE ([DOTA0,Tyr3]octreotate), DOTA-NOC ([DOTA0-
`Nal3]octreotide) and DTPA-octreotide ([DTPA0]-octreotide) were
`purchased from BioSynthema, (St Louis, MO, USA). 111InCl3 was
`purchased from Covedien (Petten, The Netherlands) and 177LuCl3
`from IDB Holland (Baarle Nassau, The Netherlands). During opti-
`misation, the typical reaction mixture for radiolabelling consisted
`of 60 MBq of 177Lu- or 111InCl3 in 0.01–0.05 M HCl with 2 nmol
`peptide dissolved in Milli-Q water, sodium acetate as buffer
`(r2 mL of 2.5 M) and 10 mL of quenchers in a final volume of
`0.14 mL (final pH 4–4.5). To inhibit oxidation and radiolysis,
`quenchers were added in various combinations and concentra-
`tions prior to radiolabelling as described in paragraph 2.4.
`Quenchers included ascorbate (Bufa BV, Uitgeest, The Nether-
`lands), gentisic acid (Tyco Health Care, Petten, The Netherlands),
`ethanol
`(Sigma-Aldrich Zwijndrecht, The Netherlands) and
`methionine (Fluka Biochemika, Switzerland). Radiolabelling of
`DOTA-TATE and other DOTA-conjugated SS-analogs with 111In or
`177Lu requires heating for 15 min at 80 1C (Breeman et al., 2005).
`DTPA-peptides were incubated for 10 min at room temperature
`(20–22 1C) (Chavatte et al., 2001). After cooling to room tempera-
`ture, quality control of DTPA- and DOTA-peptides was performed.
`Quality control includes incorporation of 111In or 177Lu as mea-
`sured by ITLC-SG (Bakker et al., 1991) and RCP of radiolabelled
`DTPA- and DOTA-peptides as measured by HPLC (de Blois et al.,
`2012). RCP of radiopeptides was determined as function of time
`post radiolabelling at room temperature at regular time intervals.
`In order to avoid false positive quality control results due to
`colloid formation (Breeman et al., 2007a; Liu and Edwards, 2001),
`quality control of DOTA-peptides was assessed after the addition
`of 5 μL 4 mM DTPA post radiolabelling (Breeman et al., 2003a,
`2003b).
`Any non-incorporated 111In or 177Lu will be rapidly captured by
`the addition of DTPA. 111In-DTPA, and 177Lu-DTPA after i.v. admin-
`istration, will be rapidly excreted via the kidneys (Breeman et al.,
`2004, 2003b).
`level (patient's dose)
`DOTA-TATE labelling at therapeutical
`was performed under the kit formulation as previously reported
`(Breeman et al., 2006) in a concentrated form (60 GBq in 3 mL).
`After QC Lu-DOTA-TATE was diluted with saline (100 mL) for
`patient infusion. We investigated the storing conditions of a
`177Lu-labelled DOTA-TATE and DOTA-NOC patient dose (3.7–
`7.4 GBq) after dilution with saline (5–100 mL)
`to maintain
`high RCP.
`
`2.2. HPLC
`
`HPLC grade methanol and trifluoroacetic acid (TFA) were
`purchased from Mallinckrodt Baker (Deventer, The Netherlands).
`All other chemicals were purchased from Sigma-Aldrich (Zwijn-
`drecht, The Netherlands). SS-analogs were analysed with a HPLC
`system (Breeze, Waters, Etten-Leur, The Netherlands), containing a
`1525 binary pump and a UV detector (W2487 Waters Dual λ
`Absorbance Detector). UV absorption was measured at 278 nm.
`A Symmetry C18 column (5 mm  4.6 mm  250 mm, Waters,
`Etten-Leur, The Netherlands) was used with a gradient profile
`as described earlier (de Blois et al., 2011), mobile phase 0.1%
`TFA (A) and methanol (B). Sample injections on the HPLC were
`performed via a Waters 717 autosampler (injection volume
`
`o200 mL). Radioactivity was monitored with a system including
`a NaI detector, digital multichannel analyzer and dedicated soft-
`ware (MetorX B.V, Goedereede, The Netherlands), connected to
`the HPLC system.
`
`2.3. Dosimetry within the reaction vial
`
`In order to investigate the influence of dose (Gy) on the
`radiolysis of SS-analogs, dose in the reaction mixture during
`the labelling procedure and storage was calculated according to
`the spherical geometry dosimetry model
`(Stabin and Konij-
`nenberg, 2000). This model derives the absorbed dose rates in
`the vials used. The complete emission spectrum of the specific
`nuclides (111In and 177Lu) was taken into account used in the
`calculations. Dose was also calculated during storage up to 7 days
`using different volumes (5, 50 and 100 mL) containing a thera-
`peutical amount (3.7 GBq) of 177Lu-DOTA-TATE.
`
`2.4. Optimizing quencher concentration
`
`To obtain maximum protection of radiolabelled SS-analogs
`and minor effect in pharmacokinetics, quencher concentration in
`reaction mixture was investigated time dependently. DOTA-TATE
`was radiolabelled with 60 MBq 111In or 177Lu in the presence of
`different concentrations of quenchers and 2 nmol of DOTA-TATE
`in a final volume of 0.14 mL. Ascorbic acid and gentisic acid were
`investigated with final concentrations of 1–20 mM, 1–50 mM for
`methionine and 2–20% (v/v) for ethanol.
`
`2.5. Radiolabelled SS-analogs in the presence of quenchers mixtures
`
`Applied quencher concentrations after optimization in reaction
`vial were: 3.5 mM for ascorbic acid and gentisic acid, 10 mM for
`methionine and 7–10% (v/v) for ethanol. Single quenchers and
`combinations of those quenchers were applied and RCP's were
`measured by HPLC up to 7 days after radiolabelling. RCP measure-
`ments were stopped when RCP decreased below 50%.
`
`2.6. Purging labelling mixture with nitrogen (N2) or oxygen (O2)
`
`To investigate any influence of O2 on the formation of radicals,
`which would lower the RCP, the reaction mixture after radiolabel-
`ling was purged for 1 h with N2 or O2 (100 mL/min). We hypothe-
`sized that N2 would decrease the O2 concentration and thereby
`In contrast, purging with O2 would
`positively influence RCP.
`increase the oxygen concentration and thereby influence RCP
`negatively. RCP was measured by HPLC up to 7 days after
`radiolabelling.
`
`2.7. Maintaining RCP and biological activity (receptor affinity)
`
`Autoradiography with stabilized 111In-DOTA-TATE (Fig. 3A) was
`performed as previously reported (de Jong et al., 2001; de Visser
`et al., 2007; Hofland et al., 1999) on rat brain sections up to 7 days
`Internalisation with stabilized 111In-DOTA-
`after radiolabelling.
`TATE was performed using CA20948 somatostatin receptor expres-
`sing cells as described previously (Bernard et al., 2000), also up to
`7 days after radiolabelling.
`
`3. Results
`
`3.1. Dosimetry within the reaction vial
`
`Dose (Gy) within reaction mixture during labelling procedure
`and storage was calculated for 111In- and 177Lu-containing vials.
`
`Evergeen Ex. 1017
`2 of 6
`
`

`

`30
`
`E. de Blois et al. / Applied Radiation and Isotopes 85 (2014) 28–33
`
`20 μM
`3.5 μM
`1 μM
`
`2
`
`4
`Time (days)
`
`6
`
`100
`
`75
`
`50
`
`25
`
`% RCP
`
`0
`
`0
`
`Fig. 2. 111In-DOTA-TATE in the presence of three different concentrations of
`ascorbic acid. X-axis shows time (days); Y-axis RCP (%). Labelling was performed
`with 60 MBq 111In and 2 nmol of DOTA-TATE in a final volume of 0.14 mL. This
`figure shows the effect of quencher concentration over time. Under same condi-
`tions, 177Lu-DOTA-TATE showed similar results.
`
`available kit formulation (OctreoScans) after reaction of their ingre-
`dients; this commercial kit contains a mixture of gentisic- and
`ascorbic acid as quenchers. Addition of ethanol to the gentisic- and
`ascorbic acid containing OctreoScans kit (see instructions for use)
`even improve stability of radiolabelled peptide (Fig. 4A and B).
`The optimal quencher mixtures as described above was tested
`within a broad range of specific activity of 111In-DOTA-TATE (50–
`190 MBq/nmol, Fig. 5A) and activity concentration (0.14–1.4 GBq/
`mL, Fig. 5B). The results were similar to those for RCP (Figs. 3 and 4).
`Fig. 6 shows the optimized storing conditions of 177Lu-DOTA-
`TATE and 177Lu-DOTA-NOC. Comparable results were obtained with
`177Lu-DOTA-TATE at a therapeutical level (3.6–7.4 GBq, see Fig. 7)
`and maintained high RCP after dilution in saline (5–100 mL).
`
`3.4. Purging labelling mixture with N2 and O2
`
`Purging the reaction mixture with N2 after radiolabelling
`resulted in 710% higher RCP after 5 days of storage. Purging with
`O2 resulted in RCP values obtained with traditional storage (data
`not shown).
`
`3.5. Maintaining RCP and biological activity (receptor affinity)
`
`In order to prove the biological activity of the radiolabelled
`SS-analogs, we studied internalisation and autoradiography during
`the 7 days of RCP monitoring (Fig. 4). Results showed a constant
`and specific binding and internalisation of SS-analogs (data not
`shown) (Breeman et al., 2007b).
`
`4. Discussion
`
`4.1. RCP quantification
`
`Since radiolysed radiopeptides often differ in charge and shape
`vs. structure of intact radiolabelled peptides, radiolysis of radi-
`olabelled peptide can be separated by HPLC and quantified by
`radiodetection. Typically, RCP of radiolabelled SS-analogs mea-
`sured by HPLC is expressed as % of radiodetected peak area
`(mV/s1) of the intact radiolabelled peptide vs. all other radio
`peaks measured during the same HPLC-analyses.
`There are no quality criteria within the field of Nuclear
`Medicine to qualify HPLC-separation methods (Breeman et al.,
`
`177Lu
`111In
`
`5 mL
`50 mL
`100 mL
`
`2
`
`4
`
`6
`
`2
`
`4
`Time (days)
`
`6
`
`5000
`
`4000
`
`3000
`
`2000
`
`1000
`
`0
`
`0
`
`8000
`
`6000
`
`4000
`
`2000
`
`0
`
`0
`
`Dose (Gy)
`
`Dose (Gy)
`
`Fig. 1. Dose calculations
`(Stabin and
`spherical geometry dosimetry model
`Konijnenberg, 2000). X-axis shows time (days); Y-axis dose (Gy). (A) Doses for 111In-
`and 177Lu-containing vials within standardised reaction mixture up to 7 days of storage.
`Reaction mixture containing 60 MBq of 177Lu- or 111InCl3 labelled SS-analog in a final
`volume of 0.14 mL. In comparison to 111In, Therapeutic nuclide 177Lu resulted in a
`5 times higher dose (Gy). (B) Dose calculations using different volumes (5, 50 and
`100 mL) were performed up to 7 days of storage for vials containing 3.7 GBq of 177Lu.
`
`Fig. 1a shows the differences is dose with a factor of 5. Fig. 1b
`shows the calculated dose during storage using different volumes
`(5, 50 and 100 mL) of saline containing a therapeutical amount
`(3.7 GBq) of 177Lu. Maximum dose was obtained (7.5 kGy) in a
`volume of 5 mL saline.
`
`3.2. Optimizing quencher concentration
`
`Quencher concentrations in reaction mixture were investigated
`time dependently by measuring RCP during storage time. Each
`quencher was investigated separately for its minimal concentration
`with maximum quenching effect (Fig. 2). Quencher mixtures were
`optimized to achieve maximum protection using these minimal
`concentrations. Under the experimental conditions the optimal
`quencher concentrations were 3.5 mM for ascorbic and gentisic
`acid, 10 mM for methionine and 10% (v/v) for ethanol. These
`concentrations had no effect on pharmacokinetics, so complete
`incorporations (499%) within described incubation time.
`
`3.3. Radiolabelled SS-analogs in the presence of quencher mixtures
`
`Reaction mixture (60 MBq 111In or 177Lu, 2 nmol DOTA-TATE in
`0.14 mL) containing ascorbic and gentisic acid (3.5 mM) and extra
`addition of ethanol clearly stabilized 111In-DOTA-TATE. Addition of
`ethanol 10% (v/v) stabilize 111In-DOTA-TATE during 7 days; higher
`concentrations (410% (v/v)) have no additional effect (Fig. 3A
`and B). We also investigated the influence of ethanol on the RCP of
`solutions of OctreoScans ([111In-DPTA0]octreotide) during storage.
`The stability of these solutions was compared with a commercially
`
`Evergeen Ex. 1017
`3 of 6
`
`

`

`E. de Blois et al. / Applied Radiation and Isotopes 85 (2014) 28–33
`
`31
`
`12.525
`
`11.538
`
`12.027
`
`12.483
`
`11.145
`
`12.537
`
`12.968
`
`12.256
`
`gentisic acid, ascorbic acid
`and ethanol
`
`12.925
`
`10.940
`
`ascorbicacid
`
`12.524
`
`12.936
`
`11.683
`
`ethanol
`
`gentisic acid, ascorbic acid and ethanol
`ascorbic acid
`gentisic acid
`ethanol
`methionine
`4x diluted
`no quencher added
`
`11.986
`
`10.676
`
`noquencheradded
`
`4xdiluted
`
`11.553
`
`13.072
`
`12.044
`
`10.707
`
`13.659
`
`11.959
`
`gentisic acid
`
`methionine
`
`100
`
`75
`
`50
`
`25
`
`0
`
`% RCP
`
`0
`
`2
`
`4
`Time (days)
`
`6
`
`Fig. 3. (A) HPLC-chromatograms of 111In-DOTA-TATE and effect of the difference quenchers and mixtures of quenchers 24 h after radiolabelling. Applied quencher
`concentrations after optimization (See paragraph optimizing quencher concentration) in reaction vial were: 3.5 mM for gentisic acid and ascorbic acid, 10 mM for methionine,
`and 7–10% (v/v) for ethanol. HPLC-chromatograms 24 h after radiolabelling can be compared in a relative way and show specific patterns of radiolysed peptide in the
`presence of different quenchers, which indicates that quenchers specifically scavenge different chemical groups and/or formed radicals (B) 111In-DOTA-TATE in the presence
`of quenchers or combinations of quenchers up to 7 days after radiolabelling. This figure shows the effect of quenchers and combination separately. The maximum error of
`inter and intra-observer was o3% and were performed nZ2.
`
`2012). Nevertheless, differences in eluents, gradient, flow, column
`type and length might result in the detection of different degrees
`of impurity, and thus variation in RCP.
`Therefore,
`in our opinion, RCP are actually expressed in
`percentage of arbitrary units (de Blois et al., 2012). Moreover,
`to enable comparison, HPLC runs were performed under standar-
`dized conditions. All HPLC-measurements were corrected for
`background. Even at low activity, the influence of background on
`calculated RCP was o3%.
`
`4.2. Addition of ethanol as a quencher
`
`Adding ethanol during or after labelling procedure could have
`severe side effects in patients with a neuroendocrine carcinoid
`tumor. Intravenous administration of a 177Lu-labelled SS-analogs
`mixture containing ethanol could lead to carcinoid syndrome
`(Adamson et al., 1971). Long et al. (1981) showed that develop-
`ment of a carcinoid syndrome could be blocked by administration
`of somatostatin. Unfortunately there is no standard maximum
`tolerated amount of ethanol which can be administered to these
`patients, since this might depend on tumour mass. A fully
`
`automated system for labelling of different SS-analogs, including
`a C18 separation using 2.5 mL of 50% of ethanol, has been applied
`in many patients world-wide, without reports of carcinoid crisis
`(Petrik et al., 2011). According to Serdons et al. (2008), radi-
`olabelled peptides can safely be used without removal of the
`ethanol after appropriate dilution with normal saline to a con-
`centration of ethanol not exceeding 10% and injection volume
`below 20 mL.
`The ready-for-use liquid formulation of the radiolabelled
`peptide allows physicians to administer
`labelled peptide
`without a radiochemical
`reaction—simply by diluting if
`necessary.
`Users still should be aware that while dilution indeed
`results in lower dose radiation, however this also decreases
`quencher concentration and thus negatively influences RCP
`during further storage. Therefore after dilution radiolabelled
`peptide should be administered as rapidly as possible to
`decrease the influence of radiolysis on radiolabelled peptide.
`To overcome this problem we recommend diluting the liquid
`formulation with quencher added to maintain a constant
`quencher concentration.
`
`Evergeen Ex. 1017
`4 of 6
`
`

`

`32
`
`E. de Blois et al. / Applied Radiation and Isotopes 85 (2014) 28–33
`
`17.916
`
`19.299
`
`19.450
`
`17.533
`
`20.671
`
`15.709
`
`ascorbic acid and
`gentisic acid
`
`ascorbic acid and
`ethanol
`
`no quencher added
`
`ethanol
`
`gentisic acid, ascorbic acid and ethanol
`gentisic- and ascorbic acid
`ethanol
`no quencher added
`
`2
`
`4
`Time (days)
`
`6
`
`100
`
`75
`
`50
`
`25
`
`% RCP
`
`0
`
`0
`
`Fig. 4. (A) HPLC-chromatograms of OctreoScans and effect of the difference quenchers and mixtures of quenchers 24 h after radiolabelling. Applied quencher concentrations
`after optimization as described in paragraph optimizing quencher concentration. HPLC-chromatograms 24 h after radiolabelling show specific patterns as well, as also
`described in Fig. 3A. The two major peaks (17.9–19.2 min) in the HPLC-chromatograms were caused by stereo isomers of the 111In-labelled DTPA (Liu et al., 2001). The two
`major pre-peaks seen after radiolysis were caused by stereo isomers of 111In-DTPA (see HPLC-chromatogram of gentisic and ascorbic acid). (B) OctreoScans in the presence of
`quenchers or combinations of quenchers. X-axis shows time (days); Y-axis % RCP. These figures show the effect of quenchers and combination up to 7 days after
`radiolabelling.
`
`0.14 GBq/mL
`0.6 GBq/mL
`1.4 GBq/mL
`
`0
`
`2
`
`4
`Time (days)
`
`6
`
`100
`
`75
`
`50
`
`25
`
`0
`
`% RCP
`
`190 MBq/nmol
`80 MBq/nmol
`50 MBq/nmol
`
`0
`
`2
`
`4
`Time (days)
`
`6
`
`100
`
`75
`
`50
`
`25
`
`0
`
`% RCP
`
`Fig. 5. 111In labelled DOTA-TATE as function of time in the presence of gentisic acid, ascorbic acid and 7% of ethanol (v/v) and the influence of specific activity (50–190 MBq/
`nmol) (A) or by decrease of the concentration of activity (0.14–1.4 GBq/mL) (B). Labelling was performed in a final volume of 0.14 mL and only the peptide amount (1–4 nmol)
`or activity (50–190 MBq) was adjusted. X-axis shows time (days); Y-axis RCP (%). Both figures show the applicability of the quencher mixture within a broad ranch of specific
`activities, thus independent of the peptide mass or activity concentration (MBq/mL).
`
`Moreover sterility is still a matter of concern, there are micro-
`biological implications for 7-day shelf-life. When the ready-for-use
`liquid formulation of the radiolabelled peptide will be used over a
`longer time period sterility should be determined before admin-
`istration, this could be performed by measuring endotoxins in final
`liquid formulation.
`
`5. Conclusion
`
`Our experiments showed that ethanol, in combination with a
`mixture of gentisic- and ascorbic acid has a superior effect in
`stabilizing radiolabelled peptides. This property enables to store
`and transport 111In- and 177Lu-labelled SS-analogs in a single-
`
`Evergeen Ex. 1017
`5 of 6
`
`

`

`E. de Blois et al. / Applied Radiation and Isotopes 85 (2014) 28–33
`
`33
`
`DTPA in [90Y-DOTA0,Tyr3]octreotide solutions containing free 90Y3þ. Nucl.
`Med. Biol. 31, 821–824.
`Breeman, W.A., Froberg, A.C., de Blois, E., van Gameren, A., Melis, M., de Jong, M.,
`Maina, T., Nock, B.A., Erion, J.L., Macke, H.R., Krenning, E.P., 2008. Optimised
`labeling, preclinical and initial clinical aspects of CCK-2 receptor-targeting with
`3 radiolabeled peptides. Nucl. Med. Biol. 35, 839–849.
`Breeman, W.A., Kwekkeboom, D.J., de Blois, E., de Jong, M., Visser, T.J., Krenning, E.P.,
`2007a. Radiolabelled regulatory peptides for imaging and therapy. Anti-Cancer
`Agents Med. Chem. 7, 345–357.
`Breeman, W.A., van der Wansem, K., Bernard, B.F., van Gameren, A., Erion, J.L.,
`Visser, T.J., Krenning, E.P., de Jong, M., 2003b. The addition of DTPA to [177Lu-
`DOTA0,Tyr3]octreotate prior to administration reduces rat skeleton uptake of
`radioactivity. Eur. J. Nucl. Med. Mol. Imaging 30, 312–315.
`Breeman, W.A.P., Melis, M.L., de Blois, E., Mueller, C., De Jong, M., Krenning, E.P.,
`2007b. Effects of quenchers on radiochemical purity and biological activity of
`[In-111-DOTA(0), Tyr(3)]-octreotate. Eur. J. Nucl. Med. Mol. Imaging 34 (Supple-
`ment: 2), S337. (S337).
`Chavatte, K., Mertens, J., Terriere, D., Van Den Winkel, P., 2001. Parameters ruling
`optimization of radiolabelling of polyamino polycarboxylated functionalized
`peptide derivatives: a case study report. Nucl. Med. Biol. 28, 745–749.
`Chen, J., Linder, K.E., Cagnolini, A., Metcalfe, E., Raju, N., Tweedle, M.F., Swenson, R.E.,
`2008. Synthesis, stabilization and formulation of [177Lu]Lu-AMBA, a systemic
`radiotherapeutic agent for gastrin releasing peptide receptor positive tumors.
`Appl. Radiat. Isot. 66, 497–505.
`de Blois, E., Chan, H.S., Konijnenberg, M., de Zanger, R., Breeman, W.A., 2012.
`Effectiveness of quenchers to reduce radiolysis of (111)In- or (177)Lu-labelled
`methionine-containing regulatory peptides. Maintaining radiochemical purity
`as measured by HPLC. Curr. Top. Med. Chem. 12, 2677–2685.
`de Blois, E., Sze Chan, H., Naidoo, C., Prince, D., Krenning, E.P., Breeman, W.A., 2011.
`Characteristics of SnO2-based 68Ge/68Ga generator and aspects of radiolabel-
`ling DOTA-peptides. Appl. Radiat. Isot. 69, 308–315.
`de Jong, M., Breeman, W.A., Bernard, B.F., Bakker, W.H., Schaar, M., van Gameren, A.,
`Bugaj, J.E., Erion, J., Schmidt, M., Srinivasan, A., Krenning, E.P., 2001. [177Lu-
`DOTA(0),Tyr3] octreotate for somatostatin receptor-targeted radionuclide ther-
`apy. Int. J. Cancer 92, 628–633.
`de Visser, M., Bernard, H.F., Erion, J.L., Schmidt, M.A., Srinivasan, A., Waser, B., Reubi, J.C.,
`Krenning, E.P., de Jong, M., 2007. Novel 111In-labelled bombesin analogues for
`molecular imaging of prostate tumours. Eur.
`J. Nucl. Med. Mol. Imaging 34,
`1228–1238.
`Erion, J.L., 2008. Use of ethanol for stabilizing a single-vial liquid formulation of a
`radiolabeled peptide. Publication no. WO2008009444. International applica-
`tion no. PCT/EP2007/006404.
`Filice, A., Fraternali, A., Frasoldati, A., Asti, M., Grassi, E., Massi, L., Sollini, M., Froio, A.,
`Erba, P.A., Versari, A., 2012. Radiolabeled somatostatin analogues therapy in
`advanced neuroendocrine tumors: a single centre experience. J. Oncol. 2012,
`320198.
`Fukumura, T., Nakao, R., Yamaguchi, M., Suzuki, K., 2004. Stability of C-11-labeled
`PET radiopharmaceuticals. Appl. Radiat. Isot. 61, 1279–1287.
`Garrison, W.M., 1987. Reaction-mechanisms in the radiolysis of peptides, polypep-
`tides, and proteins. Chem. Rev. 87, 381–398.
`Hofland, L.J., Breeman, W.A., Krenning, E.P., de Jong, M., Waaijers, M., van Koetsveld,
`P.M., Macke, H.R., Lamberts, S.W., 1999. Internalization of [DOTA degrees,125I-
`Tyr3]octreotide by somatostatin receptor-positive cells in vitro and in vivo:
`implications for somatostatin receptor-targeted radio-guided surgery. Proc.
`Assoc. Am. Phys. 111, 63–69.
`Jay-Gerin, J.P., Ferradini, C., 2000. A new estimate of the (OH)-O-center dot radical yield
`at early times in the radiolysis of liquid water. Chem. Phys. Lett. 317, 388–391.
`Jonah, C.D., 1995. A short history of the radiation chemistry of water. Radiat. Res.
`144, 141–147.
`Liu, S., Cheung, E., Rajopadhye, M., Williams, N.E., Overoye, K.L., Edwards, D.S., 2001.
`Isomerism and solution dynamics of (90)Y-labeled DTPA—biomolecule con-
`jugates. Bioconjugate Chem. 12, 84–91.
`Liu, S., Edwards, D.S., 2001. Bifunctional chelators for therapeutic lanthanide
`radiopharmaceuticals. Bioconjugate Chem. 12, 7–34.
`Liu, S., Ellars, C.E., Edwards, D.S., 2003. Ascorbic acid: useful as a buffer agent and
`radiolytic stabilizer for metalloradiopharmaceuticals. Bioconjugate Chem. 14,
`1052–1056.
`Long, R.G., Peters, J.R., Bloom, S.R., Brown, M.R., Vale, W., Rivier, J.E., Grahame-
`Smith, D.G., 1981. Somatostatin, gastrointestinal peptides, and the carcinoid
`syndrome. Gut 22, 549–553.
`Petrik, M., Knetsch, P.A., Knopp, R., Imperato, G., Ocak, M., von Guggenberg, E.,
`Haubner, R., Silbernagl, R., Decristoforo, C., 2011. Radiolabelling of peptides for
`PET, SPECT and therapeutic applications using a fully automated disposable
`cassette system. Nucl. Med. Commun. 32, 887–895.
`Schuessler, H., 1975. Effect of ethanol on radiolysis of ribonuclease. Int. J. Radiat.
`Biol. 27, 171–180.
`Serdons, K., Verbruggen, A., Bormans, G., 2008. The presence of ethanol in radio-
`pharmaceutical injections. J. Nucl. Med. 49, 2071.
`Stabin, M.G., Konijnenberg, M.W., 2000. Re-evaluation of absorbed fractions for
`photons and electrons in spheres of various sizes. J. Nucl. Med. 41, 149–160.
`Swiatla-Wojcik, D., Buxton, G.V., 2005. On the possible role of the reaction H-center
`dotþH2O-4H-2þ(center dot)OHin the radiolysis of water at high tempera-
`tures. Radiat. Phys. Chem. 74, 210–219.
`
`DOTA-TATE + 7% ethanol
`DOTA-NOC + 7% ethanol
`
`DOTA-TATE
`
`DOTA-NOC
`
`0
`
`2
`
`4
`Time (days)
`
`6
`
`100
`
`75
`
`50
`
`25
`
`0
`
`% RCP
`
`Fig. 6. Therapeutic labelling of DOTA-TATE and DOTA-NOC containing 3.7 GBq of
`177Lu. X-axis shows time (days); Y-axis in RCP (%). For patient administration,
`labelling mixtures were diluted in saline, final volume 100 mL. Under these
`conditions RCP was monitored up to 7 days after radiolabelling in the presence
`or absence of 7% of ethanol (v/v). Addition of ethanol showed a substantial
`beneficial effect on maintaining the RCP.
`
`3.7 GBq (100 mL) + 7% ethano
`3.7 GBq (5 mL) + 7% ethanol
`7.4 GBq (5 mL) + 7% ethanol
`
`3.7 GBq (100 mL)
`
`3.7 GBq (50 mL)
`
`0
`
`2
`
`4
`Time (days)
`
`6
`
`100
`
`95
`
`90
`
`85
`
`80
`
`75
`
`% RCP
`
`Fig. 7. Dilution in saline (5, 50 or 100 mL) of a therapeutic dose (3.7or 7.4 GBq) of
`177Lu-DOTA-TATE in the presence or absence of 7% of ethanol (v/v) up to 7 days
`after radiolabelling. X-axis shows time after radiolabelling (days); Y-axis in RCP (%).
`
`ready-for-use liquid formulation up to 7 days after
`vial,
`radiolabelling.
`
`Acknowledgement
`
`We would like to thank Dr. J.L. Erion for fruitful and stimulation
`discussions.
`
`References
`
`Adamson, A.R., Grahame-Smith, D.G., Peart, W.S., 1971. Pharmacological blockade of
`carcinoid flushing provoked by catecholamines and alcohol. Am. Heart J. 81,
`141–142.
`Bakker, W.H., Albert, R., Bruns, C., Breeman, W.A., Hofland, L.J., Marbach, P., Pless, J.,
`Pralet, D., Stolz, B., Koper, J.W., et al., 1991. [111In-DTPA-D-phe1]-octreotide, a
`potential radiopharmaceutical for imaging of somatostatin receptor-positive
`tumors: synthesis, radiolabeling and in vitro validation. Life Sci. 49, 1583–1591.
`Bernard, B.F., Krenning, E., Breeman, W.A., Visser, T.J., Bakker, W.H., Srinivasan, A.,
`de Jong, M., 2000. Use of the rat pancreatic CA20948 cell
`line for the
`comparison of radiolabelled peptides for receptor-targeted scintigraphy and
`radionuclide therapy. Nucl. Med. Commun. 21, 1079–1085.
`Breeman, W.A., de Blois, E., Bakker, W.H., Krenning, E.P., 2006. Radiolabeling DOTA-
`peptides with 90Y and 177Lu to a High Specific Activity, first ed. Taylor & Francis
`Group, New York.
`Breeman, W.A., de Blois, E., Bakker, W.H., Krenning, E.P., 2012. Practical aspects of
`labeling DTPA- and DOTA-peptides with 90Y, 111In, 177Lu, and 68Ga for peptide-
`receptor scintigraphy and peptide-receptor radionuclide therapy in preclinical
`and clinical applications. Monograph 16. (LESSON 5).
`Breeman, W.A., de Jong, M., de Blois, E., Bernard, B.F., Konijnenberg, M., Krenning, E.P.,
`2005. Radiolabelling DOTA-peptides with 68Ga. Eur. J. Nucl. Med. Mol. Imaging 32,
`478–485.
`Breeman, W.A., De Jong, M., Visser, T.J., Erion, J.L., Krenning, E.P., 2003a. Optimising
`conditions for radiolabelling of DOTA-peptides with 90Y, 111In and 177Lu at high
`specific activities. Eur. J. Nucl. Med. Mol. Imaging 30, 917–920.
`Breeman, W.A., De Jong, M.T., De Blois, E., Bernard, B.F., De Jong, M., Krenning, E.P.,
`2004. Reduction of skeletal accumulation of radioactivity by co-injection of
`
`Evergeen Ex. 1017
`6 of 6
`
`