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`8
`Current Radiopharmaceuticals, 2016, 9, 8-18
`
`
`Overview of Development and Formulation of 177Lu-DOTA-TATE for
`PRRT
`
`Wouter A.P. Breeman*, Ho Sze Chan, Rory M.S. de Zanger, Mark K. Konijnenberg and
`Erik de Blois
`
`Erasmus MC Rotterdam, Department of Nuclear Medicine, Dr. Molewaterplein 230, 3015 GD
`Rotterdam, Netherlands
`
`Abstract: Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin analogs has
`become an established procedure for the treatment of patients suffering from inoperable neuroendo-
`crine cancers over-expressing somatostatin receptors. Success of PRRT depends on the availability of
`the radiolabeled peptide with adequately high specific activity, so that required therapeutic efficacy
`can be achieved without saturating the limited number of receptors available on the target lesions.
`Specific activity of the radionuclide and the radiolabeled somatostatin analog are therefore important
`parameters. Although these analogs have been investigated and improved, and successfully applied for
`PRRT for more than 15 years, there are still many possibilities for further improvements that fully ex-
`ploit PRRT with 177Lu-DOTA-TATE.
`The summarized data presented herein on increased knowledge of the components of 177Lu-DOTA-TATE (especially the
`purity of 177Lu and specific activity of 177Lu) and the reaction kinetics during labeling 177Lu-DOTA-TATE clearly show
`that the peptide dose and dose in GBq can be varied.
`Here we present an overview of the development, formulation and optimisation of 177Lu-DOTA-TATE, mainly addressing
`radiochemical parameters.
`
`Keywords: 177Lu-DOTA-TATE, DOTANOC, DOTATOC, DOTA-TATE, PRRT, radiochemistry, formulation.
`
`INTRODUCTION
`Peptide receptor radionuclide therapy (PRRT) employing
`radiolabeled somatostatin analogs has become an established
`procedure for the treatment of patients suffering from inop-
`erable neuroendocrine tumors (NET) over-expressing soma-
`tostatin receptors [1-10]. The use of several radiolabeled
`peptides such as, 177Lu-DOTA-TATE (1,4,7,10-tetraaza-
`coupled Tyr3-
`cyclododecane-1,4,7,10-tetraacetic
`acid
`octreotate, Fig. 1), 177Lu-DOTATOC (DOTA coupled Tyr3-
`octreotide) and 177Lu-DOTANOC (DOTA coupled Nal3-
`octreotide) have been investigated and reported for this pur-
`pose [1-12]. The clinical results obtained with 177Lu-DOTA-
`TATE are very encouraging in terms of tumor regression.
`Also, if kidney protective agents are used, the side effects of
`this therapy are few and mild [6, 13, 14], and the median
`duration of the therapy response for these radiolabeled ana-
`logs of octreotide is 30 - 40 months [15]. The patients’ self-
`assessed quality of life increases significantly after treatment
`with 177Lu-DOTA-TATE. There is a benefit in overall sur-
`vival of several years from the time of diagnosis in patients
`treated with 177Lu-DOTA-TATE in comparison to historical
`controls (e.g. treatment with Sandostatin®) [15]. Balancing
`benefits (clinical response to radionuclide therapy) vs. risks
`(normal organ radiotoxicity) is a significant challenge [16];
`and careful assessment of biodistribution, dosimetry, and
`toxicity is thus essential, preferably on a personalized basis
`[16, 17]. The first clinical phase III study to evaluate
`
`*Address correspondence to this author at the Department of Nuclear Medi-
`cine, Dr Molewaterplein 230, 3015 GD Rotterdam, The Netherlands;
`Tel: +31 107035964; Fax: +31 107035997;
`E-mail: w.a.p.breeman@erasmsumc.nl
`
`
`
`1874-4729/16 $58.00+.00
`
`safety and tolerability of 177Lu-DOTA-TATE and compare
`therapeutic responses after 177Lu-DOTA-TATE with those
`after treatment with a high dose of the unlabeled octreotide
`analog LAR (Novartis) is currently underway in several
`countries (http://clinicaltrials.gov/ct2/show/NCT01578239?
`term=NCT01578239&rank=1) [18].
`Among other factors, success of PRRT depends on the
`availability of the radiolabeled peptide with adequately high
`specific activity (SA), so that required therapeutic efficacy
`can be achieved without saturating the limited number of
`available receptors on target lesions [19-21]. This, in turn,
`directly depends on the SA of the radionuclide and the radio-
`labeled somatostatin analog. Here we present an overview of
`the development of 177Lu-DOTA-TATE, mainly addressing
`radiochemical parameters.
`
`HISTORY OF RADIOLABELED PEPTIDES
`G-protein-coupled receptors like somatostatin receptors
`are frequently overexpressed on human tumor cells [22, 23].
`Somatostatin receptor–targeted imaging, initially with Tyr3-
`later with [111In-DTPA0]octreotide (Oc-
`octreotide and
`treoScan), was important for imaging and diagnostics of
`NET in nuclear medicine [8, 24]. Radiolabeled peptides tar-
`geting G protein–coupled receptors with DOTA as the bi-
`functional chelator were developed and have shown in vivo
`stability, favourable pharmacokinetics (PK), and high and
`specific receptor-mediated tumor uptake [8, 24-26]. The up-
`take kinetics of radiolabeled-DOTA-peptides such as DO-
`TATOC, DOTANOC and DOTA-TATE are rapid [25-27].
`These desirable PK properties are required for PRRT.
`
`© 2016 Bentham Science Publishers
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`Radiochemical Aspects on the Formulation of 177Lu-DOTA-TATE
`
`Current Radiopharmaceuticals, 2016, Vol. 9, No. 1 9
`
`OH
`
`NH
`
`O
`
`NH2
`
`NH
`
`O
`
`NH
`
`NH
`
`O
`
`O
`
`NH
`
`OH
`
`NH
`
`O
`
`NH
`
`S
`
`S
`
`NH
`
`O
`
`O
`
`COOH
`
`O
`
`O
`
`NH
`
`OH
`
`OH
`
`HOOC
`
`HOOC
`
`NN
`NN
`
`(SA) OF DOTA-
`
`DOTA-DPhe-Cys-Tyr-DTrp-Lys-Thr-Cys-Thr
`DOTA-DF-C-Y-DW-K-T-C-T
`
`Fig. (1). Structural formulae of DOTA-TATE (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid coupled Tyr3-octreotate). Molecular
`formulae, C65H90N14O19S2, molecular weight is 1435.6.
`DOTATOC, DOTANOC OR DOTA-TATE?
`These radiopeptides (DOTATOC, DOTANOC, DOTA-
`TATE) have the highest affinity to the subtype 2 of the so-
`matostatin receptor family [28], which is also most com-
`monly expressed by most NET [19, 23]. Esser et al. reported
`a longer tumor residence time for 177Lu-DOTA-TATE com-
`pared to 177Lu-DOTATOC. Despite a longer residence time
`in kidneys after 177Lu-DOTA-TATE, tumor dose will always
`be higher. Therefore, these authors concluded that the better
`peptide for PRRT is 177Lu-DOTA-TATE [28]. Wehrmann et
`al. compared the biodistribution of 177Lu-DOTA-TATE and
`177Lu-DOTANOC in patients, and concluded that tumor up-
`take and absorbed dose were comparable for both radiopep-
`tides, whereas whole-body retention was lower for 177Lu-
`DOTA-TATE, and therefore the authors advocate the use of
`177Lu-DOTA-TATE [15, 29].
`Recently Das et al. reported accumulation of 177Lu-
`DOTANOC and 177Lu-DOTA-TATE in cancerous lesions.
`Qualitative analyses of the scans showed higher retention
`and slower clearance of activity in case of 177Lu-DOTANOC
`compared to that of 177Lu-DOTA-TATE [30].
`For this overview it should also be mentioned that for di-
`agnosis of NET, 68Ga-DOTANOC has been reported to have
`the highest sensitivity and specificity, while for PRRT 177Lu-
`DOTANOC has unfavourable pharmacodynamics (PD) and
`PK [30].
`Most peptide analogs are rapidly cleared from the body
`via the kidneys and partly re-absorbed in the tubuli of these
`organs leading to a high absorbed radiation dose [31, 32]. A
`possibility to improve the results of 177Lu-DOTA-TATE or
`treatment with other radiolabeled somatostatin analogs is to
`reduce activity uptake in critical normal tissues, such as kid-
`neys [4, 5]. In clinical practice, PRRT with radiolabeled
`somatostatin analogs should always be administered with
`renal protective agents, e.g., lysine and arginine or a
`commercially available mixture of amino acids. These amino
`acids cause a reduced renal uptake of radioactivity in the
`proximal tubuli [31, 32].
`
`SPECIFIC RADIOACTIVITY
`PEPTIDES
`The SA has many different definitions, e.g. SA can be
`expressed as the activity per mass of the nuclide, or as activ-
`ity per mass of the ligand. Moreover, dimensions of SA also
`vary. As an example, activity can be expressed in Ci or Be-
`querel, or the mass in nmoles or mg. For a recent overview,
`see [33].
`There are many factors that influence the interaction of a
`radioligand with its receptor. In a saturable regulatory pep-
`tide binding processes (i.e., in vitro radioimmunoassay and
`receptor binding), the signal-to-background ratio is often
`improved by increasing the SA (expressed as activity units
`per mass units of ligand, e.g. MBq per nmol) of the ligand.
`In in vivo experiments it was shown that, contrary to what
`was expected, the percentage uptake of radiolabeled soma-
`tostatin analogs in somatostatin receptor–positive tissues is
`not optimal at the lowest dose of maximum SA; rather, the
`uptake is a bell-shaped function of the injected mass, initially
`increasing followed by a decreased uptake. These findings
`might be the result of 2 opposing effects, first a positive ef-
`fect of increasing ligand concentrations on the rate of inter-
`nalization by ligand-induced receptor clustering and sec-
`ondly a negative effect because of saturation of the receptor
`at increasing ligand concentrations [19]. This implies that the
`sensitivity of detection of somatostatin receptor-positive tu-
`mors by peptide receptor scintigraphy (PRS) might be im-
`proved by administration of an optimized dose of radio-
`ligand, as was found for other radioligands [19, 34-38].
`These findings have been confirmed in patients for [111 In-
`DTPA0]octreotide [19, 39, 40] and led to improved quality of
`imaging with a significant increase in tumor uptake.
`Jonard et al. [41] presented data on tissue distribution af-
`ter the administration of 86Y-DOTATOC, labeled with vari-
`ous amounts of DOTATOC (range of 50-500 (cid:1)g); with
`higher peptide amounts the kidney dose was not affected,
`however, tumor dose decreased. Velikyan et al. also investi-
`gated the impact of peptide mass on binding to NET soma-
`tostatin receptors in vivo by using 68Ga-DOTATOC as tracer
`at a constantly high SA, preceded by injection of 0, 50, 250,
`or 500 (cid:1)g of octreotide (Sandostatin; Novartis), administered
`
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`Breeman et al.
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`atom% of177Lu
`
`6
`
`0
`
`4
`2
`t½ of 177Lu (= 6.71 days)
`
`Fig. (2). Atom% of 177Lu as f(t(cid:2)) of 177Lu. Atom% are expressed
`as % of 4070 GBq per mg Lu (4070 GBq per mg Lu is theoretical
`maximum, see Table 1), or 5.13 *10-11 moles per 37 MBq 177Lu
`(see Table 1 and in text above SA of 177Lu).
`applied for optimizing personal patient peptide dose in
`PPRT. SA of 177Lu and 177Lu-DOTA-TATE are therefore
`important radiochemical and clinical parameters and are ad-
`dressed separately, in SA of 177Lu, SA of 177Lu-DOTA-
`TATE and FAQ’s.
`SA OF 177LU
`As mentioned earlier, SA has many different definitions
`[45, 46]. In theory 1 nmol of a DOTA-peptide can incorpo-
`rate 1 nmol Lu3+, this number indicates the maximal theo-
`retical SA of 177Lu-DTPA- or DOTA-peptides, see Table 1.
`The highest achievable SA of radioligands, e.g. 177Lu -
`DOTA-TATE can be radiolabeled in theory at a level of 0.72
`GBq 177Lu per nmol ligand (see Table 1). However, there are
`several factors influencing SA, such as 177Lu from (n, (cid:3)) re-
`actor-produced from enriched 176Lu contains 175Lu and 176Lu,
`and in variable amounts. For recent overviews, consult Refs
`[47-50]. The presence of 176Lu reduces the maximally
`achievable SA in practice to 0.12 GBq 177Lu per nmol ligand
`(see Table 1). 177Lu from (n, (cid:3)) reactor-produced from en-
`riched 176Yb has a higher SA, and revealed a higher maximal
`achievable SA: 0.42 GBq 177Lu per nmol DOTA-TATE [33]
`(see Table 1).
`Another possibility to express SA of 177Lu is in atom%,
`expressed as % of theoretical value 4070 GBq per mg Lu, or
`5.13 *10-11 moles per 37 MBq 177Lu (see Table 1). In Fig. (2)
`the atom% of 177Lu as f(t(cid:2)) of 177Lu are shown. To illustrate
`this, suppose SA of 177Lu is 100 atom%, after 1 half-life of
`177Lu the activity has decreased to 50%, whereas the corre-
`sponding mass has decreased also 50%, thus the ratio hasn’t
`changed and remains 100 atom%. Fig. (2) also shows an-
`other frequently encountered misunderstanding, e.g. suppose
`a SA of 177Lu of 80 atom% (e.g. from ORNL, see legend of
`Table 1 and [51]), after 1 half-life SA hasn’t decreased to 40
`atom%. Indeed, the activity halved, but the mass of Lu has
`changed also.
`To illustrate and clarify this, after 1 half-life the atom%
`has decreased to 67 atom%, after 2 half-lives to 50 atom%
`etc. (Fig 2). Thus in contrast to 40 and 20 atom%, resp., that
`is frequently suggested.
`In short, correction for the transformation of 177Lu to
`177Hf is required, and, in addition, the SA of 177Lu is higher.
`
`10 Current Radiopharmaceuticals, 2016, Vol. 9, No. 1
`
`Table 1. Physical characteristics and constants from reactor-
`produced 177Lu from enriched 176Lu (n, (cid:1)) 177Lu.
`
`Target
`
`Decay product of 177Lu
`
`t(cid:2) [days]
`
`nmoles per GBq 177Lu
`pmoles per 37 MBq 177Lu
`
`Ci 177Lu per mg
`GBq per mg 177Lu
`
`Maximal achievable SA of 177Lu-DOTA-
`peptide [GBq.nmol-1]
`
`in Theory
`
`in Practice
`
`176Lu
`
`177Hf
`
`6.71
`
`1.39
`51.3
`
`110
`4070
`
`
`
`0.72a
`
`0.12b,
`0.42c
`0.5d
`
`a: Since, in theory 1 nmol of a DOTA-peptide can incorporate 1 nmol nuclide, this
`number indicates the maximal theoretical SA of 177Lu-DOTA-peptides
`b: data from (n, (cid:1)) reactor-produced 177Lu from enriched 176Lu [19].
`c :177Lu reactor-produced via (n, (cid:1)) from enriched 176Y [86, 87]. In theory, the SA of
`this 177Lu is 0.72 GBq per nmol, however in practice, the highest achieved and reported
`SA was 0.42 GBq 177Lu per nmol DOTA-peptide [33] (see also SA of 177Lu-DOTA-
`TATE)
`d: at high thermal neutron flux (e.g. 1.5*1015 neutrons cm-2 s-1) as in the High Flux
`Isotope Reactor at Oak Ridge National Laboratory (ORNL), after 4 days of irradiation
`80% of all the Lu atoms can be in the form of 177Lu [47, 51]. DOTA-TATE was suc-
`cessfully radiolabeled with this material, up to a SA of 0.5 GBq nmol-1 [33].
`Table is adapted from Ref [33].
`
`10 minutes before the tracer [42]. Nine patients with gastro-
`enteropancreatic NET were included. Accumulation of activ-
`ity in the tumors varied and depended on the total amount of
`the pre-administered octreotide. In 5 of 6 patients, the high-
`est tumor- to-normal tissue ratio was found when 50 (cid:1)g of
`octreotide was preadministered. Thus again, optimizing mass
`improved image contrast. However, 1 patient showed a con-
`tinuously increasing tumor uptake even with higher octreo-
`tide pre-administered. The application of 68Ga-labeled ligand
`for optimizing therapeutic applications of concordant radio-
`therapeutic labeled ligand needs further dosimetric studies. A
`relation (such as in PK and clearance) between the ligands
`labeled with 68Ga versus the therapeutic radionuclide (e.g.
`90Y or 177Lu) at early time points also needs to be established
`[42].
`Beauregard et al. suggested that tumor sequestration of
`68Ga-DOTA-TATE is a major factor leading to a sinkeffect
`that decreases activity concentration in healthy organs such
`as the kidney. Compared with a fixed-dose PRRT protocol,
`an adjusted-dose regimen tailored to tumor burden, body
`habitus and renal function may allow greater radiation dose
`to individual lesions without substantially adding to toxicity
`in normal tissues [43]. On the other hand, Kletting et al. pre-
`fers to avoid the introduction of unnecessary inaccuracy in
`dosimetry, and therefore recommended using the same sub-
`stance along with the same amount for pretherapeutic meas-
`urements and therapy [44].
`From the above-mentioned arguments it can be con-
`cluded that the highest SA does not always result in the
`highest target uptake, and the amount of administered radio-
`activity and the amount of ligand is a potent tool and can be
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`Radiochemical Aspects on the Formulation of 177Lu-DOTA-TATE
`
`Current Radiopharmaceuticals, 2016, Vol. 9, No. 1 11
`
`To illustrate the high SA of ORNL-produced 177Lu was con-
`firmed as the highest achieved SA of 177Lu -DOTA-TATE
`was 0.5 GBq per nmol DOTA-TATE.
`
`plexed and rerouted in vivo effectively the specification for
`the % of incorporation (measured by ITLC) was lowered to
`97 % at our Institution [57].
`
`SA OF 177LU-DOTA-TATE
`In daily practice 177Lu -DOTA-TATE is produced at a
`SA of 40 MBq per nmol. Unfortunately, the need for high
`SA is often compromised by conflicting practical parame-
`ters, such as the pH and solubility of the radionuclide during
`radiolabeling. The pH determines reaction rates and yields,
`i.e. the rate of formation of the metal-DOTA complexes in-
`creases with pH, but on the other hand the solubility of Lu3+
`decreases when pH is increased [52]. Moreover, reaction
`kinetics differ for each radionuclide and reactions can be
`hampered by contaminants, including contaminants from
`target material and decay products, see Table 1 [20]. Fortu-
`nately, 177Hf4+ (decay product of 177Lu, see Table 1) does not
`interfere with the incorporation of 177Lu in the DOTA-
`moiety under these conditions [21]. Eventually the highest
`achievable SA of 177Lu-DOTA-TATE is determined by the
`SA of 177Lu (Table 1) [46].
`It should also be noticed that the specifications men-
`tioned on the datasheet of vendors frequently state that metal
`ions like Zn and Fe will not exceed 20 (cid:1)g per Ci 177Lu (1 Ci
`177Lu equals 5.13*10-8 moles, see Table 1), however, when it
`reaches this level, and expressed in molar ratio vs. Lu, it
`would be 12 and 7 times higher, respectively, and this will
`certainly affect the highest achievable SA of 177Lu-DOTA-
`TATE.
`
`LABELING OF LU-DOTA-TATE AND QUALITY
`CONTROL
`A typical reaction mixture for radiolabeling is 37 GBq (1
`Ci, for 4 patients) 177LuCl3 in 1 mL 0.05 M HCl with 1 mg
`DOTA-tate in 2.5 mL 50 mM sodium-ascorbate and gentisic
`acid and a final pH of 4 [38, 53-55]. Reaction kinetics for
`labeling DOTA-peptides differ per radionuclide, e.g. 177Lu,
`reactions at pH 4–4.5 were completed after 20 min at 80°C
`[20]. After radiolabeling and cooling the reaction mixture to
`room temperature a chelator, such as DTPA is added. There
`are several reasons for this addition. First, it is difficult to
`take a representative sample from a solution containing
`DOTA-conjugated analogs labeled with radionuclides that
`are known to form colloids. For example, in the accurate
`determination of unchelated 177Lu during the standard quality
`control by ITLC (0.1 M Na-citrate, pH 5 as mobile phase) or
`HPLC, the unchelated will be rapidly bound to the origin of
`the ITLC or to HPLC column [56]. This will result in a false
`identification of the incorporation or RadioChemical Purity
`(RCP), respectively [56], see Quality Control by HPLC,
`below. The addition of a chelator solves this problem, and
`the addition is therefore necessary (see RCP and Quench-
`ers, below). Second, the free ionic fraction of radionuclide in
`radiolabeled DOTA-peptides can effectively be complexed
`by the addition of chelator in vitro, and this results in an effi-
`cient complexation of the free ionic fraction of radionuclide
`and excretion as such [57]. Since the free ionic fraction of
`radionuclide in radiolabeled DOTA-peptides can be com-
`
`QUALITY CONTROL BY HPLC
`Since radiolysis products of radiopeptides often differ in
`charge and shape vs. structure of the intact radiolabeled pep-
`tide, radiolysis of radiolabeled peptide can be quantified by
`HPLC. Typically RCP of radiolabeled DOTA-peptides is
`measured by HPLC and expressed as % of radiodetected
`peak area (e.g. (cid:1)V.sec-1) of the intact radiolabeled peptide vs.
`all radio peaks measured during the same HPLC-analyses
`[33, 58]. There are reports on the determination of peaks by
`HPLC, including accuracy, linearity, precision, repeatability
`and detection limit [33, 58]. To our knowledge, there are no
`criteria to qualify a HPLC separation method plus radiode-
`tection in the field of nuclear medicine as perfect, good or
`good enough. Therefore, we suggested a set of standardized
`requirements to quantify RCP by HPLC for radiolabeled
`DTPA- or DOTA-peptides, including a base-to-base separa-
`tion of metal-DOTA-peptide vs. DOTA-peptide [33, 58].
`In our opinion, RCP values are currently expressed in
`Arbitrary Units. The requirements to standardize RCP meas-
`urements would open standardization to compare RCP quan-
`tifications between different systems and laboratories.
`The following items on Quality Control (QC) are items to
`(cid:3)enable and optimize(cid:3) intra- and inter-laboratory compari-
`sons of QC of 177Lu-DOTA-TATE:
`i.
`ITLC is for monitoring incorporation of the radionuclide
`ii.
`ITLC cannot replace HPLC.
`iii. Radiodetection and software for determination of peak
`areas are currently not standardised.
`In addition, incorporation is not identical to RCP, thus
`ITLC is not a correct technique to monitor RCP.
`Asti et al. [59] reported base-to-base chromatographic
`separations by UPLC (Ultra HPLC) of DOTA-TATE labeled
`with different non-radioactive metal ions. How this new
`chromatographic technique will affect radiodetection (e.g.
`balance between sensitivity of the detector and resolution by
`HPLC and UPLC) is currently under investigation.
`1. The tools in analytical chemistry are constantly improv-
`ing and applied in nuclear medicine: e.g. we are now
`able to quantify peptide content and purity of DOTA-
`TATE and other DOTA-peptides [45], including,
`2. Quantification and identification of metal impurities
`already present in the DOTA-moiety of DOTA-TATE
`and other DOTA-peptides [45], and to
`3. Quantify SA of 177Lu [45] and eventually.
`4.
`Improve SA of the radioligand.
`
`RCP AND QUENCHERS
`Measuring and quantification of RCP is not standardized,
`and therefore comparison of radiolabeling and RCP of regu-
`
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`latory peptides between different HPLC-systems and be-
`tween laboratories, is cumbersome. De Blois et al. presented
`an overview in measuring and quantification of radiolysis
`RCP of radiolabeled regulatory peptides, including 177Lu-
`DOTA-TATE [58]. To calculate the radiation dose in the
`reaction vials during radiolabeling and storage of the radio-
`peptides, a dosimetry model was developed. With this model
`RCP in the absence of quenchers can now be predicted and
`the effects of quenchers studied [58, 60, 61].
`The conclusion was that maintaining high RCP requires a
`combination of quenchers [33, 58, 60, 61].
`It would be desirable to radiolabel, store and transport a
`ready-for-use one-vial liquid formulation (for PRS and
`PRRT) of radiolabeled peptides. The use of ethanol, in com-
`bination with a mixture of gentisic- and ascorbic acid, has
`superior effects on stabilizing radiolabeled somatostatin ana-
`logs [60]. As a consequence, 177Lu-DOTA-TATE can now
`be stored and transported in a single-vial ready-for-use liquid
`formulation up to 7 days after radiolabeling.
`Although not fully within the scope of this “Overview of
`the development, formulation and optimising of 177Lu-
`DOTA-TATE, mainly addressing radiochemical parame-
`ters”, there are several practical items addressed here which
`are strongly related with the application of 177Lu-DOTA-
`TATE: 1st is the trend of applying robotics for radiolabeling
`and, 2nd is the 177mLu-containing waste.
`
`APPLICATION OF ROBOTICS
`Maus et al. investigated the current trend to include a C18
`solid phase extraction (SPE) post-radiolabeling in order to
`remove unwanted components such as HEPES (4-(2-
`hydroxyethyl)-1-piperazineethanesulfonic acid) and non-
`incorporated 177Lu from the injection solution [62]. How-
`ever, with the introduction of SPE purification, quenchers
`such as gentisic acid and ascorbic acid were also removed
`from the injection solution. As a result, there was a concor-
`dant dramatic drop of the RCP of 177Lu-DOTA-TATE. Maus
`et al. therefore concluded that re-addition of ascorbic acid
`post C18 SPE purification is required to maintain the RCP of
`177Lu-DOTA-TATE [62].
`
`177MLU-CONTAINING WASTE
`As in most cases in Nuclear Medicine departments radio-
`active waste streams are based on the half-lives of the used
`radionuclides, e.g. waste containing 177Lu is mixed with 131I-
`containing waste.
`The level of clearance of radioactive waste is country-
`and t(cid:1)-dependent, e.g. in European Union, the clearance
`level of radionuclides with t(cid:1) > 100 days is 10 Bq per g.
`However, within the European Union there are countries
`with more restrictive levels: e.g. 1 Bq per g. It is obvious, the
`reduction of the clearance level of radionuclides (10 (cid:2) 1 Bg
`per g) will take an extra 3-4 half- lives of 177mLu (480-640
`days) of storage to reach that level of 1 Bg per g. As an ex-
`ample, suppose the 177mLu activity is 0.01% of the 177Lu ac-
`tivity. After 14 half-lives of 177Lu (± 13 weeks) 177mLu activ-
`ity equals the 177Lu activity. The ratio in activity of 177mLu
`vs. 177Lu at the end of irradiation depends, among others on
`
`Breeman et al.
`
`the irradiation time [48, 63]. 177mLu content from reactors
`such as HFR in the Netherlands and BR2 in Belgium is 0.05
`kBq 177mLu per MBq 177Lu (0.005% [64] and <0.05% (ac-
`cording to specifications for GMP-produced 177Lu, IDB,
`Baarle Nassau, the Netherlands), and 0.015% from the
`Dhruva reactor (Mumbai, India)[6].
`The presence of 177mLu in 177Lu should not be ignored,
`therefore Bakker et al. advised to collect high-activity 177Lu-
`and 177mLu-containing waste separately [64].
`
`FUTURE ASPECTS
`Although DOTA-peptides can be labeled with therapeu-
`tic radionuclides at high SA, the SA (expressed as activity
`per mass of ligand), may be too low for PRS or PRRT. In
`short, delivery of sufficient amounts of radioactivity to these
`targets may not be high enough for PRS or PRRT. There are
`various reasons for this, e.g. the amounts of available recep-
`tor is too low (receptor density in tissue is in the range of 10-
`13 and 10-9 M [21, 65-68]. There may be several other ways
`to circumvent this limitation, such as different ways of ad-
`ministration influencing PK of the radioligand, such as long-
`lasting infusions of the radioligand, fractionating the dose or
`combinations hereof [40, 69, 70] intra-arterial [71-73] or
`intratumoral administration [18, 74].
`Studies in patients have thus far been performed with
`somatostatin receptor agonists (DTPA-octreotide, DOTA-
`TOC, DOTANOC, and DOTA-TATE), because such ago-
`nists are internalized in the (tumor) cells and radioactivity is
`retained in the cell. Another approach is the use of an an-
`tagonist of the ligand [75-78]. Receptor antagonists are not
`internalized and, therefore, thought to be inappropriate for
`imaging and therapy, as we reported for DTPA- and DOTA-
`bombesin agonists [35]. However, ligands labeled with
`short-lived radionuclides might be possible, especially with
`(cid:1)-emitters [71, 72, 79-81], since these radionuclides have a
`high Linear Energy Transfer (high energy deposition within
`a short range), consequently the cell kill probability is high,
`but only if the target (e.g. DNA) is within range. For a recent
`overview, consult other sources [18, 43].
`An important item for the success of PRRT is implement-
`ing knowledge from radiobiology, like the research on radio-
`sensitivity of tumor (and within types of tumor) and normal
`tissue. Moreover, there is a myriad of combinations, includ-
`ing pharmacological options, such as the tyrosine kinase
`inhibitor Sunitinib [82], mTOR inhibitor Everolimus [83],
`and a variety of combinations of chemotherapeutics such as
`Capecitabine and Temolozomide in pancreatic NET [17, 84].
`With all the above-mentioned possibilities in mind and
`although radiolabeled somatostatin analogs have been inves-
`tigated and successfully applied for PRRT for more than 15
`years, there are still many possibilities to improve and fully
`exploit PRRT with 177Lu-DOTA-TATE, as discussed in de-
`tail in Refs [8, 10, 18, 73].
`
`FAQ’s
`
`Hofman and Ricks recently raised the question whether
`PRRT with 177Lu-DOTA-TATE should be performed under
`standardized, randomized or personalized conditions? [17].
`
`Evergeen Ex. 1027
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`
`

`

`Radiochemical Aspects on the Formulation of 177Lu-DOTA-TATE
`
`Current Radiopharmaceuticals, 2016, Vol. 9, No. 1 13
`
`Table 2. FAQ 3 represents a typical patient dose of 177Lu-DOTA-TATE and contains 7.4 GBq 177Lu at a SA of 740 GBq per mg Lu
`with 140 nmoles (= 200 (cid:1)g) DOTA-TATE. FAQ 4-7 are a summary of the variation in DOTA-TATE, Lu-DOTA-TATE
`and 177Lu-DOTA-TATE as f(GBg, SA and nmoles). Details are described in FAQ 3-7, see also Fig (3).
`
`FAQ
`
`177Lu in GBq
`
`SA of 177Lu in GBq per mg Lu
`
`DOTA-TATE in nmoles
`
`Lu-DOTA-TATE in nmolesa
`
`177Lu-DOTA-TATE in nmoles
`
`Atom% of 177Lu in Lub
`
`3
`
`7.4
`
`740
`
`140
`
`57
`
`10
`
`18
`
`4
`
`7.4
`
`740
`
`100
`
`57
`
`10
`
`18
`
`bratio 177Lu vs. Lu
`a10 (cid:5)g Lu equals 57 nmoles Lu and 1 GBq 177Lu equals 1.39 nmoles 177Lu (see Table 1)
`Atom% is defined and discussed earlier in SA of 177Lu, see also Fig (2)
`b Ratio of SA of 177Lu (in GBq per mg Lu) vs. 4070 (= theoretical max SA of 177Lu, also in GBq per mg Lu)
`
`
`0.18
`
`0.18
`
`FAQ 3
`
`5
`
`3.7
`
`370
`
`100
`
`57
`
`5
`
`9
`
`0.09
`
`6
`
`7.4
`
`1110
`
`100
`
`38
`
`10
`
`27
`
`0.27
`
`7
`
`11.1
`
`1110
`
`100
`
`57
`
`15
`
`27
`
`0.27
`
`FAQ 4, reduction in DOTA-TATE
`
`FAQ 6, reduction in DOTA-TATE and
`50% increase in SA 177Lu
`FAQ 7, reduction in DOTA-TATe and
`50% increase SA 177Lu and in dose
`
`150
`
`100
`
`50
`
`0
`
`nmoles
`
`DOTA-TATE
`
`Lu-DOTA-TATE
`
`Lu-DOTA-TATE
`
`
`
`177
`Fig. (3). Illustrates the content of a typical patient dose of 177Lu-DOTA-TATE, expressed in nmoles of DOTA-TATE, Lu-DOTA-TATE and
`177Lu-DOTA-TATE (FAQ 3).
`
`From the above-mentioned arguments, which include in-
`creased knowledge including insight on SA of 177Lu and
`177Lu-DOTA-TATE, use of 177Lu after end of irradiation and
`the atom% of 177Lu as ƒ(t), more parameters became avail-
`able to vary the dose in GBq and in mass of the ligand. See
`also Specific Radioactivity (SA) of DOTA-peptides.
`To illustrate the potentials of radiochemistry to personal-
`ize PRRT with 177Lu-DOTA-TATE a summary of FAQ’s on
`dose in GBq and mass in nmoles were formulated, discussed
`and explained in Table 2 and Fig. (3).
`1. What is the highest achievable SA of 177Lu-DOTA-
`TATE ?
`
`2. What is the optimal amount of DOTA-TATE for PRRT?
`3. Which part of DOTA-TATE is Lu-DOTA-TATE and
`177Lu-DOTA-TATE?
`4. Which part of DOTA-TATE is 177Lu-DOTA-TATE
`when the mass of DOTA-TATE is lowered?
`5. Which part of DOTA-TATE is 177Lu-DOTA-TATE
`when the SA of 177Lu is increased by 50%?
`6. Which part of DOTA-TATE is 177Lu-DOTA-TATE
`when the dose in MBq is decreased by 50%?
`
`Evergeen Ex. 1027
`6 of 11
`
`

`

`14 Current Radiopharmaceuticals, 2016, Vol. 9, No. 1
`
`7. Which part of DOTA-TATE is 177Lu-DOTA-TATE
`when the dose in MBq is increased by 50%?
`
`FAQ 1. What is the highest achievable SA of 177Lu-
`DOTA-TATE ?
`Initially 177Lu-DOTA-peptides were labeled at a molar
`ratio of peptide vs. Lu of (cid:1) 4 (range from 4 up to > 1000).
`Whereas, as a result of a decade of investigations on DOTA-
`TATE with natLu and 177Lu and concordant reaction kinetics
`of natLu-DOTA-TATE and 177Lu-DOTA-TATE, we are now
`able to label DOTA-TATE vs. Lu at a molar ratio of 1.05
`with full (>99%) incorporation of 177Lu. With the (n,(cid:4)) pro-
`duced 177Lu the highest achievable SA in practice is mainly
`the resultant of the grade of enrichment of target, thermal
`flux and length of irradiation time. For reviews see [47-50,
`63, 85]. As spin off of this research we could improve the
`SA of 177Lu-DOTA-TATE [45, 46]. Eventually the highest
`achievable SA of 177Lu-DOTA-TATE is determined by the
`SA of 177Lu (see Table 1, details on maximal achievable SA
`in the legend (b-d) of Table 1).
`
`FAQ 2. What is the optimal amount of DOTA-TATE for
`PRRT?
`The optimal amount of DOTA-TATE for PRRT depends
`on several items, including
`a.
`the “available receptors” and
`b. when "enough" activity is targeted for successful PRRT.
`The activity that can be administered depends on the
`corresponding SA of 177Lu and the amount of DOTA-
`TATE.
`A typical dose of DOTA-TATE is 140 nmoles (equals
`200 (cid:1)g DOTA-TATE) and incorporates 7.4 GBq 177Lu at a
`SA of 177Lu of 740 GBq per mg Lu.
`In the following FAQ’s variation in dose, either in mass
`of DOTA-TATE, or in GBq or in SA of 177Lu is presented
`(Table 2 and Fig. 3) to illustrate the effects on these varia-
`tions.
`
`FAQ 3. Which part of DOTA-TATE (in terms of mol%)
`is Lu-DOTA-TATE and 177Lu-DO