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9. LUTETIUM-177 PRODUCTION AND RADIOPHARMACEUTICALS

9.1. INTRODUCTION

Lutetium-177, though a relatively late entrant to the radionuclide therapy, is one of the most promising radionuclides for targeted therapy. Although the interest in 177Lu by the nuclear medicine community is a recent development, the US patent ‘Organic amine phosphonic acid complexes for the treatment of calcifi c tumors’ fi led by the DOW Chemical Company in 1987 and based on the University of Missouri-Columbia work on 153Sm-EDTMP covered 177Lu, 159Gd, 166Ho and 175Yb in addition to 153Sm [1]. The fi rst publication on the use of 177Lu for radiopharmaceuticals development was by Keeling et al. in 1988 on the uptake of 177Lu on hydroxyapatite particles [2]. A series of papers, fi rst in the year 1991 [3], were published on the radiolabeling of CC-49, a murine monoclonal antibody that recognized the tumor associated glycoprotein 7 (TAG-72), with 177Lu for the development of a radioimmunotherapy agent.

In 1994, Balasubramanium reported the production of no carrier added 177Lu by irradiating natural ytterbium [4]. Ando et al. reported the preparation of 177Lu-EDTMP [5] which was followed by a similar report by Solla et al. which also contained details about human administration of the radiopharmaceutical [6]. In 2001, Kwakkeboom et al. reported the preparation of 177Lu-DOTATATE and its clinical use in patients [7]. Liu et al. reported the synthesis of a 177Lu labeled vitronectin receptor antagonist peptide, RGD, in the same year [8]. The author started working on 177Lu in 1999 and published the fi rst paper on the labeling of 177Lu with polyaminophosphonate ligands as potential agents for bone pain palliation in 2002 [9]. As technical offi cer of the International Atomic Energy Agency (IAEA), the author was responsible for two Coordi-nated Research Projects (CRPs), the objectives of which were to enhance the production of 177Lu and the development of 177Lu radiopharmaceuticals [10]. The CRPs enhanced the development and wider application of 177Lu based radiopharmceuticals.

The application of 177Lu as a therapeutic radionuclide has widened since then and targeted therapy using 177Lu radiopharmaceuticals is one of the fastest growing branches of therapeutic nuclear medicine. The author’s contributions in the development of 177Lu radiopharmaceuticals reported in his various pub-lications are detailed in this chapter [9, 11-18].

Apart from the favorable radionuclidic characteristics such as low energy β– emitter with a favorable half-life of 6.73 days, the most important aspect

185

which makes 177Lu an attractive radionuclide for targeted therapy is the very high thermal neutron capture cross-section of the target 176Lu (176Lu(n,γ)177Lu (σ = 2060 barns)). The cross-section is the highest encountered among all (n,γ) produced radionuclide presently used for therapy. The large cross-section of the reaction ensures that 177Lu can be produced with adequately high specifi c activity for radionuclide therapy applications even while using moderate fl ux reactors. The high cross-section also ensures that there will be no constraints in the large scale production of 177Lu and hence the increasing global demand for the radionuclide in the coming years can be met comfortably. In the long run, the cost of the radionuclide also should come down signifi cantly with the entry of more producers into the market. The long half-life of 177Lu provides logistic advantage for facilitating supply to places far away from the reactors.

9.2. INORGANIC CHEMISTRY OF 177Lu

Lutetium is commonly mentioned as the last member of the lanthanide series. However, it could be also considered as the fi rst element of the d-block in the 6th period because it has completely fi lled 4f orbital containing all the 14 elec-trons. In its +3 oxidation state (the most stable oxidation state), Lu has empty s, p and d orbitals and a closed-shell of f orbitals. Electrons in f orbitals are incapable of bond formation as these electrons are tightly bound due to high effective nuclear charge and are not infl uenced by ligands surrounding the metal ion. Thus, the chemistry of lutetium is mostly governed by the empty s, p and d orbitals. The Lu+3 is a hard Lewis acid. Due to the completely fi lled f orbital, the ionic radius of Lu3+ is the smallest among the lanthanides and as a consequence the numbers of ligands that may be placed around the ion are limited. The coordination number is mostly dictated by the reciprocal repulsions between the various ligands without any relevant infl uence attributable to the orbitals involved in bond formation (s, p and d). Lu forms stable complexes with several ligands and coordination numbers of 6, 7, 8 and 9 are reported. Lutetium forms highly stable, nine coordinate, DOTA (1,4,7,10-tetraazacyclo-dodecane-N,N,N’’,N’’’-tetraacetic acid) complex with a stability constant of about 25.4 and hence is a preferred chelate for carrying the radionuclide in most radiopharmaceuticals [19].

Naturally occurring lutetium has one stable isotope 175Lu (97.41%) and one long-lived beta active isotope, 176Lu, with a half-life of 3.78×1010 years (2.59% natural abundance). Besides these two isotopes, more than fi fty radionuclides have been produced including 23 nuclear isomers ranging in mass number from 150 to 184.

186

9.3. RADIONUCLIDIC CHARACTERISTICS OF 177Lu

Lutetium-177 decays with a half-life of 6.73 days and β– emissions of Eβ(max) 498 keV (78.6%), 385 keV (9.1%), 176 keV (12.2%). There are also two gamma rays of energy 208 keV (11.0%) and 112 keV (6.4%). The β– particles emitted by 177Lu have moderate tissue penetration thereby making the radionu-clide suitable for targeting small tumors and metastasis without giving much radiation dose burden to the non-target tissue. The two gamma photons emitted are suitable for imaging by single photon emission computed tomography (SPECT). This is an important aspect, not available with pure β– emitters such as 32P, 89Sr and 90Y, since access to pharmacokinetic data by serial scintigraphic imaging offers an important opportunity to estimate the individual tumor radiation dose.

9.3.1. 177Lu: A METALLIC ANALOGUE OF 131IRadionuclide therapy owes its existence largely to 131I and its favorable prop-erties as a therapeutic radionuclide. Apart from the favorable radionuclidic characteristics, its long half-life and ease of production in large quantities in a nuclear reactor has helped its wide availability across the world. The ability of 131I to accumulate in human thyroid in large amounts resulted in its use for the treatment of hyperthyroidism and thyroid cancer eventually making 131I the most successful therapeutic radionuclide. Lutetium-177 can be considered as a metallic analogue of 131I and could be used instead of 131I wherever the in vivo uptakes mechanism allows. Table 9.1 gives a comparison of the radionuclidic characteristics and production methods between 131I and 177Lu. Table 9.1. Comparison of the properties of 131I and 177Lu

Characteristics Iodine-131 Lutetium-177Half-life 8.02 d 6.73 d

Beta energy (%) 608 keV (89.9%)330 keV (7.27%)250 keV (2.1%)

498 keV (78.6%) 385 keV (9.1%)176 keV (12.2%)

Gamma energy (%) 636 keV (7.2%)364 keV (81.7%)284 keV (6.14%)

113 keV (6.4%)208 keV (11%)

Production 235U (n,f) 131Te(n,γ)131Te to 131I

176Lu(n,γ)177Lu176Yb(n,γ)177Yb to 177Lu

Specifi c activity Up to ~20% isotopic abundance. 129I and 127I present

Up to ~70% (direct) and 100% (indirect) isotopic abundance.

177mLu presentChemistry Halogen Metal chemistry (+3)Use (main) Targeted therapy

(mainly thyroid and others)Targeted therapy

(other than thyroid)

187

Lutetium-177 has a half-life of 6.73 days which is comparable to the 8.02-day half-life of 131I. Hence, the duration of dose delivery using 177Lu will be similar to that of 131I once the radiopharmaceutical is taken up by the tissue.

Lutetium-177 emits β– particles of slightly lower energy as compared to 131I. Hence, the tissue penetration is lower than that of 131I, which is a favorable feature in some applications such as treating metastatic diseases. Low penetra-tion also helps to reduce the damage to the normal tissues by cross fi re effect [20], at the same time killing the cancer cells which are in the vicinity even if there is no accumulation of the radionuclide in the diseased cell.

The major problem encountered with the use of 131I for therapy is the high abundance (~95%) of gamma rays associated with its decay which is not use-ful for therapy, but gives unnecessary radiation dose to the organs other than the target organ. The patients may suffer tremendous psychological impact due to their unavoidable isolation in wards which is necessary to prevent the public from receiving unnecessary dose from the activity injected into the patient. Lutetium-177 also has two gammas but due to their lower energy and abundance (113 keV, 6.4% and 208 keV, 11%), the external radiation dose will not be very high. At the same time, these low energy, low abundant gammas can be used for imaging and tracking the path of the radiopharmaceutical within the body. The data collected from such imaging can be used for calculating patient spe-cifi c dosimetry.

Iodine-131 can be produced either by irradiation of natural tellurium in a nuclear reactor, through the reactions 130Te(n,γ)131Te decaying (T1/2 = 25.0 min) to 131I, or by nuclear fi ssion. Natural Te has three isotopes 126Te, 128Te and 130Te, and all these isotopes also capture neutrons with the simultaneous production of 127I (inactive) and 129I (T1/2 = 5x105 y). The presence of the inactive and long-lived radioiodine reduces the specifi c activity of 131I signifi cantly; and only about 20% of the total iodine atoms present with as 131I and this will keep reducing once the production is over. Though enriched 130Te can be used for the production of 131I, it is not considered a feasible alternative due to increase in cost of production. Iodine-131 is one of the major fi ssion products when 235U or other heavy elements undergoes fi ssion with fi ssion yields of ~6%. Hence, 131I could also be isolated as a byproduct during the processing of 99Mo through the fi ssion route (fi ssion Moly). During nuclear fi ssion 127I and 131I are also formed and the achievable isotopic abundance is only ~20%. However, the specifi c activity of 131I obtained using natural tellurium target or fi ssion is con-sidered adequate for all applications including antibody labeling for which it is used. Likewise, high specifi c activity 177Lu (with isotopic abundance > 20% could be prepared by irradiating highly enriched 176Lu in high fl ux research reactors. No carrier added (NCA) 177Lu can be prepared by the indirect route starting with 176Yb.

Due to the close similarities in the nuclear properties of 131I and 177Lu, as well as few added advantages of 177Lu, the 177Lu is emerging as a preferred

188

isotope for applications where 131I was used earlier. The exception will be the continued use of 131I for thyroid related applications and for radiolabeling small molecules where DOTA conjugation is a problem.

9.4. PRODUCTION LOGISTICS OF 177Lu [12]

The production logistics of 177Lu for different applications and the advantages/disadvantages associated with different production procedures are discussed in a paper published by the author [12]. Lutetium-177 can be prepared by direct or by indirect route and the specifi c activity achievable in both the methods are very high for most applications. Both the direct and indirect route has its own advantages some of which will be elaborated in the following sections.

9.4.1. DIRECT ROUTE FOR PRODUCTION OF 177LuThe direct route of production via 176Lu(n,γ)177Lu, has a very high nuclear reac-tion cross-section (σ = 2065 barns) and, as mentioned earlier, it is the highest cross-section encountered in the production of a therapeutically useful radio-nuclide. The abundance of 176Lu in natural target is 2.6% and highly enriched (> 80%) 176Lu targets are available at reasonable cost. The use of enriched targets could provide a specifi c activity of up to 70 Ci/mg (theoretical specifi c activ-ity 109 Ci/mg) resulting in isotopic abundance > 60% while using very high fl ux research reactors [21, 22]. A specifi c activity of ~20 Ci/mg is found adequate for radiolabeling of peptides and antibodies [23].9.4.1.1. Optimization of irradiation timeCareful optimization of irradiation time is essential in order to obtain the high-est specifi c activity of the 177Lu produced. In high fl ux reactors the target burn up will be considerably high due to the high thermal neutron capture cross- -section of 176Lu and hence, in this case, the usual assumption that the number of target atoms remains constant during the period of irradiation will not be valid. Considering the number of target atoms is a function of irradiation time, the commonly used differential equation

dN2/dt = N1σφ – N2λ can be modifi ed as

dN2/dt = N0e–σφtσφ – N2λ where, N0 – number of 176Lu atoms used as target (at t = 0), N1 – number of 176Lu atoms at any time t, N2 – number of 177Lu atoms at any time t, λ – decay constant of 177Lu, σ – thermal neutron capture cross-section of 176Lu, φ – ther-mal neutron fl ux of the reactor, t – time of irradiation. The 177Lu activity produced at the end of bombardment can be calculated using the following equation, which is obtainable by solving the modifi ed differential equation mentioned above.

189

N0λσφA = ⎯⎯⎯⎯ [e–σφt – e–λt] (λ – σφ)

Depending on the neutron fl ux the activity of 177Lu produced will be the maximum after certain duration of irradiation, beyond which the activity will decrease owing to the high target burn up (Fig. 9.1). Higher the thermal neutron

fl ux of the reactor, shorter will be the time of irradiation needed for attaining maximum activity. Therefore, in order to obtain maximum specifi c activity, the time of irradiation must be judiciously optimized according to the neutron fl ux available. It is quite likely that at very high neutron fl ux and long irradiation the target may be completely burned as reported at the SM2 reactor (a fl ux of > 2x1015 n cm–2 s–1 and irradiation cycle of 21 days) [24]. In such cases it will be even worth putting low enriched target as partial enrichment will take place during irradiation. 9.4.1.2. Contribution from double neutron captureIf natural or low enriched Lu is used as the target, 175Lu present in the natural target will undergo successive neutron capture contributing to the 177Lu activ-ity produced. Figure 9.2 shows the 177Lu activity produced from 175Lu and 176Lu while using natural Lu target for 7-day irradiation at different thermal neutron fl uxes. For comparison, 177Lu activity obtainable from 100% enriched 176Lu target is also given. Although, the 177Lu activity produced by double neutron capture of 175Lu is insignifi cant at relatively low neutron fl ux, the contribution from this route becomes quite signifi cant with the increase of neutron fl ux. This is because of the fact that the activity of a radionuclide produced by successive

Fig 9.1. 177Lu activity produced as a function of irradiation time at different neutron fl ux

190

neutron capture process is proportional to the square of the neutron fl ux. There-fore, the specifi c activity of 177Lu obtainable from high fl ux reactor using natural Lu target will be much higher than that expected from 176Lu(n,γ)177Lu only as part enrichment of the target takes place during irradiation.9.4.1.3. Co-production of 177mLu as a radionuclidic impurityThe direct route of 177Lu production also results in the formation of some amount of 177mLu, a long-lived (T1/2 = 160.5 d) metastable radionuclide of lutetium. The presence 177mLu in a radiopharmaceutical preparation, though projected as one of the drawbacks in the direct method, is not a problem for the use of the ra-dionuclide in patients. The 177mLu being chemically same as 177Lu, has the same biological properties and hence will not increase the radiation dose to tissues other than the target organs. This is in contrast to conventional radionuclidic impurities which invariably increases the radiation dose to non-target organs. The presence of 177mLu has been considered as a problem in certain countries to manage the waste arising out of the nuclear medicine department and hence these countries are increasingly pushing the use of 177Lu produced through the indirect route.

9.4.2. INDIRECT ROUTE FOR PRODUCTION OF 177LuThe indirect route of production can be used for the preparation of NCA 177Lu. The nuclear reaction for the production of 177Lu is shown below.

Fig. 9.2. 177Lu activity produced with natural and enriched Lu target at different thermal neutron fl ux (irradiation time 7 days)

191

β– β–176Yb(n,γ)177Yb ⎯⎯⎯⎯→ 177Lu ⎯⎯⎯⎯→ 177Hf (stable)

σ = 2.85 barns T1/2 = 1.9 h T1/2 = 6.71 dFrom theoretical calculations employing the appropriately modifi ed form

of the Bateman equation, it can be shown that irradiation of 99% enriched 176Yb2O3 target at a reasonably high thermal neutron fl ux of 5×1014 n cm–2 s–1 will produce ~60 mCi/mg (of Yb target) which is no carrier added and hence

will have the theoretical specifi c activity of 109 Ci/mg (of Lu). However, the radiochemical processing adapted in the indirect method has to be extremely neat as the presence of the target material 176Yb in the 177Lu will reduce the apparent specifi c activity of the product as both Lu and Yb have identical reac-tion kinetics with most of the known ligands used, including DOTA.

Table 9.2. 177Lu activity produced via direct and indirect routes (irradiation at 5×1014 n cm–2 s–1)

Duration of irradiation (d)

177Lu yield (mCi)Indirect route

(per mg of 100% 176Yb target)Direct route

(per mg of 100% 176Lu target)1 10.9 8 936 3 20.6 22 815 7 29.7 35 852 14 57.0 36 666 21 84.7 27 704 28 104.7 18 740

Fig. 9.3. Comparison of 177Lu activity produced via direct and indirect routes

192

The production of 177Lu through the indirect route will signifi cantly increase the cost of the radionuclide as the production involves the use of enriched target and an elaborate radiochemical processing is needed. Large quantities of 176Yb enriched target will be needed for the preparation of 177Lu in usable quantities for therapy. The use enriched targets with low activation cross-section is not economical for isotope production, as a signifi cant part of the target will be wasted or has to be carefully recovered.

Table 9.2 gives a comparison of the production yields of 177Lu through direct and indirect route at a neutron fl ux of 5×1014 n cm–2 s–1. The data is graphically presented in Fig. 9.3. As the amount of radionuclide produced is signifi cantly lower, the reactor utilization cost for the indirect route of produc-tion will be much higher.

9.5. PRODUCTION OF 177Lu [9]

Lutetium-177 suitable for certain applications, such as bone pain palliation, which do not require high specifi c activity can be prepared by using natural lutetium targets. However, it is advantageous to use enriched targets for 177Lu as enriched 176Lu with isotopic abundance above 80% is available relatively inexpensive (200 euros per mg) from multiple sources.

9.5.1. IRRADIATION AND RADIOCHEMICAL PROCESSING Typically, about 6 mg of natural Lu2O3 powder (spectroscopic grade, > 99.99% pure) sealed in an aluminum can is irradiated in Dhruva reactor for 5-7 days at a thermal neutron fl ux > 3×1013 n cm–2 s–1. The irradiated target was transferred to a glass container inside a lead-shielded plant and dissolved in 1 M HCl by gentle warming. The resultant solution was evaporated to near dryness and reconstituted in double distilled water. A known aliquot was drawn from the stock for assessment of radioactivity content and evaluating the radionuclidic purity.

For production of high specifi c activity 177Lu, isotopically enriched Lu2O3 (60.6% 176Lu) (Isofl ex, USA) was irradiated. A stock solution of enriched target was prepared by dissolving 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 fl ame sealed ampoule was then placed inside an aluminum can, sealed by cold welding and irradiated. The can was irradiated at a thermal neutron fl ux of > 3×1013 n cm–2 s–1 for 5-7 days. The chemical processing of the irradiated target was carried out following the same procedure described for natural Lu2O3 target.

Radioactivity was assayed 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 the γ ray spectrum

193

of the appropriately diluted solution after radiochemical processing using an HPGe detector (EGG Ortec/Canberra detector) connected to a 4K multichannel analyzer (MCA) system. A 152Eu reference source (Amersham Inc.) was used for both energy and effi ciency calibration. Several spectra were recorded for each batch at regular time intervals. Samples initially assayed for 177Lu were preserved till 177Lu activity is signifi cantly decayed (over 10-15 T1/2 of 177Lu, i.e. for a period of 2-3 months) and re-assayed to determine the activity of long- -lived 177mLu (T1/2 = 160.5 d). Appropriately diluted sample solutions were counted for 1 h.

9.5.2. PRODUCTION YIELDSThe typical yields of 177Lu from natural as well as enriched targets for different durations of irradiation in Dhruva reactor (3×1013 n cm–2 s–1) are shown in Table 9.3. These values were in excess of theoretically calculated values at that time, which are also given alongside in Table 9.5. This difference is attributed to the contribution from epithermal neutrons (resonance integral = 1087 b), which was

not accounted in theoretical calculations. The discrepancies in the practical yields and theoretical yields were further explained in a paper published by Dvorakova et al. based on their studies on the production of 177Lu at the FRM-II reactor [25]. The variations in the yield of 177Lu between different batches were mainly at-tributed to the fl uctuations in the irradiation conditions such as the exact dura-tion, intervening shutdown and variation of neutron fl ux due to the variation in power level of reactor operation.

9.5.3. RADIONUCLIDIC PURITYThe radionuclidic purity of 177Lu produced from either natural or enriched target was near 100% as estimated by the γ ray spectrum. In a typical γ ray spectrum of the irradiated target, after chemical processing, the major γ peaks observed were 72, 113, 208, 250 and 321 keV, all of which corresponded to the

Table 9.3. Specifi c activities of 177Lu produced from natural and enriched Lu2O3 target by thermal neutron bombardment at nutron fl ux of ~3×1013 n cm–2 s–1

Target Duration of irradiation (d)Specifi c activity (at EOB)

(TBq·g–1)theoretical experimental

Natural Lu 3 1.50 2.5 ± 0.35 2.27 3.3 ± 0.27 2.90 4.0 ± 0.3

60.6% enriched Lu 3 34.96 72 ± 55 52.91 92 ± 37 67.59 110 ± 5

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photopeaks of 177Lu. This was further confi rmed by monitoring peak area values at those peaks which reduced according to the half-life of 177Lu.

As discussed earlier, 177mLu (T1/2 = 160.5 d) is co-produced upon thermal neutron bombardment of Lu2O3 target. However, the γ ray spectrum of the ir-radiated Lu target after chemical processing did not show any peak of signifi -cant intensity corresponding to the photopeaks of 177mLu (128, 153, 228, 378, 414, 418 keV). Trace level of 177mLu activity present was assayed later, by re-cording γ ray spectrum of the initial sample aliquot with high radioactive concentration, after allowing suffi cient cooling to decay of 177Lu activity. The average level of burden in 177Lu due to 177mLu was found to be 150 nCi per mCi or ~1.5x10–2% back calculated to EOB.

9.6. REVIEW OF 177Lu RADIOPHARMACEUTICALS

There are no registered or approved 177Lu radiopharmaceuticals at present. How-ever, there are a few products which are in use in humans. The most widely used 177Lu tracer, 177Lu-DOTATATE, is emerging as a successful radiopharma-ceutical for the treatment of neuroendocrine tumors. There are a few other products which can be developed into successful radiopharmaceuticals. Fol-lowing is a brief review of the current status of 177Lu radiopharmaceuticals.

9.6.1. 177Lu LABELED MONOCLONAL ANTIBODIESThe similarity in the radionuclidic characteristics of 131I and 177Lu initiated the use of the latter for radiolabeling of monoclonal antibodies. Table 9.4 gives a list of monoclonal antibodies labeled with 177Lu and biologically investigated.

There are several studies describing the radiolabeling of monoclonal anti-bodies with 177Lu for the development of therapeutic radiopharmaceuticals and at least three products were administered in humans and their effi cacy evalu-ated. The fi rst report was the radiolabeling of CC49, a murine monoclonal antibody specifi c to tumor associated antigen 72 (TAG-72) which reacts with a wide range of human carcinomas. A Phase I clinical trial involved the treat-ment in combination with taxol, a radiosensitizer [29]. Four of the 17 patients with disease measurable with CT had a partial response whereas 4 out of 27 patients with non-measurable disease had progression free intervals ranging from 18 to 37 months. A Phase I/II clinical trial with 27 chemotherapy resistant ovarian cancer patients treated with 177Lu-CC49 showed antitumor effects of the radiopharmaceutical [30]. Long-term survival was seen in fi ve patients with microscopic diseases. Other patients with gross disease showed varying re-sponse.

177Lu labeled tetravalent single chain Fv construct (Sc(Fv)2) of CC49 monoclonal antibody was prepared and comparative biodistribution, blood clearance and tumor targeting with 177Lu-CC49 was studied in athymic mice

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Antibody Targeting antigen Type of cancer Clinical status Reference

CC49 Tumor associated antigen (TAG-72)

Colon, ovarian, adeno carcinoma etc.

Phase 1/II clinical trial in ovarian cancer

[3, 26-36]

CC49 single chain Fv construct

Tumor associated antigen (TAG-72)

Several cancers such as colon, ovarian and adeno

carcinoma

Nil [37, 38]

CC49 single chain Fv construct

(pretargeting)

Tumor associated antigen (TAG-72)

Colon, ovarian, adeno carcinoma etc.

Nil [39]

J591 Prostate specifi c membrane antigen

Prostate cancer Clinical trial [40-43]

Anti-CD20rituximab

CD20 Non-Hodgkin’s lymphoma 2 patients studied

[44]

Cetuximab EGFR Several targets No [45]

Pertuzumab HER-2, tyrosine kinase receptor

Breast cancer and others [46, 47]

huA33 Antigen 33 (A33) Colorectal cancer No [48]

Anti-L-CAM L1 cell adhesion protein

Neuroblastoma, renal and ovarian

No [49, 50]

Antitenacin (ch81C6)

tenacin Brain tumour No [51]

Anti-VEGF (VG76e)

Vascular endothelial growth factor

[52]

MOv18 α-isoform of folate receptor

Prostate cancer No [53]

7E11 PSMA Prostate cancer No [54]

hu3S193 PSMA Prostate cancer No [55]

Trastuzumab (Herceptin®)

HER-2 Breast cancer No [56]

MAb U36 CD44v6 No [57]

mAb cG250 RCC Renal cell carcinoma No [58]

Epratuzumab (hLL2)

CD22 Non-Hodgkin’s lymphoma No [59]

RS7 Epithelial glycoprotein Small cell lung carcinoma No [60]

Table 9.4. Monoclonal antibodies labeled with 177Lu and studied for radioimmuno-therapy

196

bearing tumor xenografts [36]. The product showed less renal uptake, fast clearance but with equivalent tumor uptake with respect to177Lu-CC49. Pretar-geting approach by administering CC49 single chain Fv construct linked to streptavidin followed by the administration 177Lu-DOTA-biotin 4 h later was also suggested [37].

A clinical trial with J591, a monoclonal antibody against prostate specifi c membrane antigen (PSMA) labeled with 177Lu is reported in patients suffering from prostate cancer [40-43]. The studies provided important information on the potential for 177Lu-J591 in therapy of hormone refractory prostate cancer. However the effi ciency of this therapy in metastatic bone cancer need to be determined in Phase II clinical trials.

In a recent study, anti-CD20 antibody (rituximab) labeled with 177Lu was administered in two patients with relapsed non-Hodgkin’s lymphoma. The administration of 177Lu-DOTA-rituximab was tolerated well and 177Lu-rituximab could be a good agent for the treatment with few side effects for patients with relapsed NHL [44].

Radiolabeling of cetuximab, a monoclonal antibody against epidermal growth factor (EGFR) with 177Lu through different bifunctional chelating agents and the in vitro stability studies done were reported [45]. The PET images after co-injection of 89Zr and 177Lu antibody conjugates showed similar biodistribu-tion.

Pertuzumab is a monoclonal antibody against HER-2, a tyrosine kinase receptor that is overexpressed in several carcinomas, especially breast cancer. The 177Lu labeled pertuzumab showed favorable targeting properties in BALB/c mice with HER-2–overexpressing xenografts [46, 47]. The absorbed dose in tumors was fi ve times higher than the absorbed dose in blood and more than seven times the absorbed dose in any other normal organ. Experimental therapy showed that 177Lu-pertuzumab delayed tumor progression compared with con-trols.

Monoclonal antibody A33 (huA33) is a potential targeting agent against colorectal carcinoma since the A33 antigen is expressed in > 95% of all colorectal cancers, both primary tumors and metastases. The antibody, huA33 was labeled with 177Lu and biodistribution studies were carried out in nude mice with colorectal SW1222 tumor xenografts. The tumor uptake of 177Lu-huA33 showed highly favorable biodistribution, with high tumor uptake, indicating that the conjugate may be suitable for radioimmunotherapy of colorectal cancer [48].

The L1 cell adhesion protein is overexpressed in tumors, such as neuro-blastomas, renal cell carcinomas, ovarian carcinomas, and endometrial carci-nomas, and represents a target for tumor therapy with anti-L1-CAM antibody chCE7. The 177Lu-DOTA labeled anti-L1-CAM antibody was evaluated in vitro [49]. Divalent fragments of this internalizing antibody were also labeled with 177Lu and the tumor and tissue uptake in nude mice with SK-N-BE2c xenografts were evaluated [50].

197

Chimeric antibody antitenascin (ch81C6) labeled with 177Lu was tested in athymic mice bearing subcutaneous D54 MG human glioma xenografts [51].

Vascular endothelial growth factor (VEGF) is one of the molecules which regulate angiogenesis. The VG76e, an anti-VEGF monoclonal antibody, was labeled with 177Lu and its biodistribution and gamma camera imaging was done [52].

The mouse monoclonal antibody MOv18, directed against the α-isoform of folate receptor (FR), was labeled with 177Lu and its pharmacokinetics and long-term therapeutic effi cacy were studied in a xenografted mouse model. The 177Lu-MOv18 was able to eradicate small size tumor masses expressing the antigen [53].

The monoclonal antibody 7E11, is used in ProstaScint (111In-7E11) an ap-proved immunodiagnostic agent. The 177Lu-7E11 which showed uptake and retention in mice bearing prostate tumor, hence, could be a radioimmunothera-peutic agent for prostate cancer [54].

Lutetium-177 labeled anti-Lewis Y monoclonal antibody (hu3S193) was studied in mice bearing prostate cancer xenografts. The in vivo biodistribution and tumor localization of 177Lu-hu3S193 was evaluated in mice bearing estab-lished DU145 tumor xenografts and the results of the biodistribution studies demonstrated specifi c targeting of DU145 prostate cancer xenografts by the agent. The 177Lu-hu3S193 caused specifi c and dose-dependent inhibition of prostate cancer tumor growth [55]. The monoclonal antibody, trastuzumab used in treating breast cancer, was labeled with lutetium-177 thorough DOTA chelator and in vitro quality control tests were performed as a fi rst step in the production of a new radiopharmaceutical [56]. Immunoreactivity and toxicity of the complex were tested on MCF7 breast cancer cell line. At a concentration of 1.9 nM, 90 ± 5% of the cells were killed. The results are promising for further evaluation in animals and possibly in humans as a new radiopharmaceutical for use in radioimmunotherapy against breast cancer. In a preliminary study, the chimeric monoclonal antibody MAb U36 specifi c to the antigen CD44v6 was labeled with 177Lu and uptake, retention, and affi nity were determined [57].

Chimeric antirenal cell cancer monoclonal antibody G250 (mAb cG250) was radiolabeled with 177Lu and biodistribution and therapeutic effi cacy were studied in nude mice bearing SK-RC-52 human RCC xenografts. In radioim-munotherapy (RIT) experiments at maximum tolerated dose, tumor growth was delayed most effectively by cG250 labeled with 177Lu than with 90Y and 186Re labeled antibodies and was found superior to that of 131I-cG250. The radio-nuclides 177Lu and 90Y led to higher radiation doses to the tumor and most likely are better candidates than conventionally radiolabeled 131I for RIT with cG250 in patients with renal cell carcinoma (RCC) [58].

The monoclonal antibody hLL2 (epratuzumab), a humanized mAb which internalizes and directed against the CD22 antigen, was labeled with 177Lu and

198

administered in nude mice with subcutaneous human lymphoma xenografts. Tumor uptake of 177Lu-hLL2 was higher than tumor uptake of 131I- and 186Re-hLL2 [59].

Tumor targeting and therapeutic effi cacy of 177Lu labeled monoclonal anti-body RS7 (antiepithelial glycoprotein-1) was evaluated in a human non-small cell lung carcinoma xenograft model [60]. Therapy was performed in nude mice with subcutaneous Calu-3 xenografts using the maximal tolerated dose

(MTD) of 177Lu-DOTA-RS7 (10.2 MBq (275 μCi)). Complete regression of the tumors, with an initial mean tumor volume of 0.24 cm3, was observed with 177Lu-DOTA-RS7 doses ranging from 5.6 MBq to 9.3 MBq (150-250 μCi) per nude mouse, with no signifi cant difference in response rate noted between the doses in this range. The conclusion of the studies was that 177Lu-RS7 is an ef-fective radioimmunoconjugate for radioimmunotherapy.

9.6.2. 177Lu LABELED PEPTIDES Bioactive peptides labeled with 177Lu constitute a group of clinically promising radiopharmaceuticals. Major advantages of peptides as vehicles for carrying radionuclides are their rapid pharmacokinetics including fast diffusion and target localization combined with rapid excretion of the unbound peptides. Peptides can be modifi ed by employing well established synthetic procedures without compromising their biologic affi nity. Unlike antibodies, peptides are non-immunogenic. Advantages of peptides compared to other small molecular weight targeting agents are that they are more tolerant to chemical modifi cation essential for radiolabeling and optimizing pharmacokinetic properties. A prac-tical advantage in the preparation of peptide based radiopharmaceuticals is that

Table 9.5. Peptides labeled with 177Lu and studied for peptide receptor radionuclide therapy

Peptide receptor Receptor Tumor type ReferenceTATE SSTR 2 Neuroendocrine tumors [65-71]TOC SSTR 2, 3 and 5 Neuroendocrine tumors [72-76]

Lanreotide SSTR Neuroendocrine tumors [13]NOC SSTR Neuroendocrine tumors [77]

Bombesin/GRP BB1-4, BB2=GRP SCLC, colon, breast, glioblastoma, prostate

[78-83]

Bombesin/AMBA GRP SCLC, colon, breast, glioblastoma, prostate

[84-89]

RGD integrin Tumors exhibiting neoangiogenesis [6, 90]α-MSH α-MSH Melanoma [91-93]

Substance P NK1 Glioblastoma, astrocytoma, MTC, breast cancer, intra- and

peritumoral blood vessels

[94]

199

peptides can be synthesized conveniently using automated modules readily available in the market. Table 9.5 gives the peptides and their targets which are used for the development of radiopharmaceuticals.

Most prominent class of radiolabeled peptides explored in radiopharma-ceuticals chemistry is the ones used for targeting somatostatin receptors. The targeting with somatostatin analogous exploit the property of cells of intestinal adenocarcinomas, lymphomas and other neuroendocrine tumors to express higher concentrations of somatostatin receptors in comparison to that of normal tissues. These somatostatin peptide analogues are disulfi de linked cyclic octapep-tides. There are fi ve subtypes of somatostatin receptors (sst 1-5) and the peptide analogues synthesized have varying affi nity towards the individual subtypes. For example, octreotide (D-pheala-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH) binds to somatostatin receptor subtypes (sst 2 and sst 5). A few agents based on somato-statin peptides labeled with other radionuclides such as 111In, 68Ga, 90Y etc. are already in clinical use for diagnosis as well as for therapy [61-64]. Two of the 177Lu-radiopharmaceuticals, [177Lu-DOTA-Tyr3]-octreotate (177Lu-DOTATATE) [65-71] and [177Lu-DOTA-Tyr3]-octreotide (177Lu-DOTATOC) [72-76], have shown highly encouraging results in terms of tumor regression in patients suf-fering from different types of neuroendocrine tumors which are identifi ed to overexpress the respective receptor subtypes. The DOTATATE has a nine--fold higher affi nity for the somatostatin receptor subtype 2 as compared with DOTATOC and therefore it is expected to be more potent for carrying out targeted therapy in patients suffering from some form of neuroendocrine tumors. Other peptide analogues studied are DOTA-lanreotide [14] and DOTANOC DOTA0,[1-Naphthylanaline]3-octreotide (DOTANOC) [77].

Apart from the somatostatin analogues, bombesin is one of the widely investigated peptide studied for radiolabeling with 177Lu [78-83]. Bombesin, a fourteen amino acid peptide was originally isolated from frog skin. It binds to gastrin releasing peptide (GRP) receptors and is a good tumor marker for small cell carcinoma of lung, gastric cancer and neuroblastoma. A bombesin analogue, AMBA (DOTA-CH2CO-G-4-amino-benzoyl-Q-W-A-V-G-H-L-M-NH2) was also used for radiolabeling with 177Lu [84-89]. The 177Lu-AMBA showed improved pharmacokinetics and higher retention of radioactivity in the tumor. Another peptide extensively studied for 177Lu labeling is RGD, a vitronectin receptor antagonist binding to integrin (αvβ3) receptors [7, 90]. Other peptides studied for 177Lu labeling are α-melanocyte stimulating hormone (α-MSH) [91-93], substance P [94] and folate [95]. Substance P, a highly biologically active pep-tide which is easily degraded in vivo, has high affi nity for NK-1 receptors. These receptors are expressed on glioma tumor cells and tumor vessels.

9.6.3. 177Lu BONE PAIN PALLIATION AGENTSThe low energy β– particles of 177Lu are ideally suited for bone pain palliation. The 177Lu-EDTMP was fi rst reported by Ando et al. [5] and Solla et al. who

200

performed limited preclinical human study with this agent [6]. Systematic screening of the 177Lu complexes of several cyclic and acyclic polyaminophos-phonate ligands were done by the author and his colleagues [9, 11]. The ability of the cyclic ligand, DOTMP to form 177Lu complex at lower ligand : metal ratio tempted the detailed biological evaluation of 177Lu-DOTMP [11, 96-98]. How-ever, considering the fact that 153Sm-EDTMP is an approved product, 177Lu-EDTMP was preferred for an IAEA sponsored clinical trial. Detailed biological studies of 177Lu-EDTMP was carried out in order to collect preclinical data needed to start a Phase I/II clinical trial in patients suffering from bone metastasis due to prostate cancer [17, 18].

9.6.4. 177Lu LABELED PARTICULATESAs mentioned in the beginning, 177Lu-hydroxyapatite (HA) particle was the fi rst 177Lu based product to be investigated [2]. The 177Lu-HA particles were further investigated and their biological properties were found desirable for radiation synovectomy [99] and not so favorable for potential application in the treatment of liver cancer [100].

9.6.5. 177Lu SMALL MOLECULESSome of the other interesting reports are the preparation of 177Lu labeled estra-diol [16], sanazole [101] and porphyrin [102] as potential cancer targeting agents. Investigation of 177Lu labeled E. coli heat stable enterotoxin for target-ing uroguanylin receptors on human colon cancer is also reported [103].

9.7. DEVELOPMENT OF 177Lu BASED RADIOPHARMACEUTICALS

In the fi rst research coordination meeting of an IAEA CRP ‘Labeling techniques of biomolecules for targeted radiotherapy’, held in 1998 at the European Insti-tute of Oncology, Milan, the author suggested the inclusion of 177Lu as one of the potential radionuclides to be explored for the development of therapeutic radiopharmaceuticals. The work on 177Lu radiopharmaceuticals was started by the author at the Bhabha Atomic Research Centre in the year 1999 and part of the work carried out is published in the fi nal report of the above CRP [104]. The following sections give details towards the development of 177Lu radio-pharmaceuticals.

9.7.1. DEVELOPMENT OF 177Lu-PHOSPHONATES FOR BONE PAIN PALLIATION As mentioned in the introduction, Ando et al. fi rst reported [5] 177Lu-EDTMP as a potential tracer for bone pain palliation and also, as reported by Solla et al., few patients received 177Lu-EDTMP injection in Argentina [6]. The author once

201

started working with 177Lu, realized the immense advantage of using 177Lu based radiopharmaceuticals for bone pain palliation, especially, as 177Lu can overcome some of the disadvantages of the existing bone pain palliation agents.9.7.1.1. Limitations of existing bone pain palliation agents There are three registered radiopharmaceuticals for bone pain palliation of which 153Sm-EDTMP (Quadramet®) and 89SrCl2 (Metastron®) are the most widely used. Though 153Sm can be prepared in adequate quantities in medium fl ux reactors, the short half-life (47 h) is a major disadvantage for transportation of the radiopharmaceutical due to signifi cant loss of activity during transit. It is also necessary to prepare large quantities of activity to allow for decay losses during production and transport of the radiopharmaceutical.

The major problem with 89SrCl2 is the limited production capacity of 89Sr, due to the very low neutron capture cross-section of 88Sr, making this product expensive and unavailable for majority of the patients. The third product, 186Re-HEDP (hydroxy ethylidine diphosphonate) is not widely used as rhenium chemistry is diffi cult and hence the radiochemical purity of the product needs to be closely monitored. Unbound 186Re, as perrhenate, is taken up by the thyroid which needs to be blocked prior to administration of perchlorate. There is also need to have very highly enriched target to make 186Re of high radionuclidic purity.9.7.1.2. Advantages of 177Lu based bone pain palliation agents Lutetium-177 has several advantages to be used for radionuclide therapy as listed below:

The physical half-life of • 177Lu is 6.7 days, which provides logistic advantage for production, distribution and quality assurance. The • 177Lu is produced by (n,γ) activation of 176Lu and has an extremely high nuclear reaction cross-section (2065 barns). Hence, large quantities of 177Lu can be produced in the low/medium fl ux research reactors available in many countries. There are more than 35 research reactors worldwide having fl ux values of more than 1x1014 n cm–2 s–1 that can be used for production of 177Lu [105]. The • 177Lu production capacity can be signifi cantly increased by irradiating enriched targets in high fl ux research reactors. The • 177Lu decays by emission of β– particles with Eβ(max) of 497 keV (78.6%), 384 keV (9.1%) and 176 keV (12.2%) and the low energy β– particles are better suited for bone pain palliation as it will have lesser bone marrow involvement compared to all the three registered bone pain palliating agents. The • 177Lu also emits γ photons (113 keV, 6.4%, 208 keV, 11%), suited for gamma camera imaging and quantitation.

9.7.1.3. Screening of acyclic and cyclic phosphonates for 177Lu labeling and bone uptake studies [9, 11]The 153Sm-EDTMP was selected by the Missouri group as the most appropriate candidate as bone pain palliating agent after extensive radiochemical and bio-

202

evaluation of several 153Sm agents [106]. The affi nity of the metal coordinated phosphonate ligands for calcium in actively growing bones is considered to be the factor responsible for their selective localization into metastatic lesions. The 153Sm-EDTMP is a well accepted radiopharmaceutical for pain palliation due to skeletal metastases and several thousands of patients have received this radiopharmaceutical.

Cyclic phosphonates are capable of forming complexes at lower ligand to metal ratios and with better thermodynamic stability and kinetic inertness as documented in the case of 166Ho-DOTMP [107]. Thermodynamic stability of the metal based radiopharmaceuticals is an important aspect as the dissociation of the radiometal from the chelate in vivo may result in the accumulation of radioactivity in non-target organs. Sm(III) chelates of DOTMP are reported to have higher thermodynamic stability over the corresponding acyclic analogue EDTMP [108]. Kinetic inertness also plays a signifi cant role for the in vivo stability of a metal chelate. While fast dissociation kinetics is characteristic of lanthanide metal complexes of acyclic chelators, complexes containing macro-cyclic chelators are expected to be more kinetically inert.

The chemistry of Lu varies signifi cantly from Sm and other lanthanides due to its difference in ionic size as well as the completely fi lled f orbital. Hence, it was decided to carry out a systematic screening of cyclic as well as acyclic polyphosphonates for radiolabeling with 177Lu and study the in vivo behavior of the respective complexes to develop 177Lu based radiopharmaceu-ticals [9, 11]. 9.7.1.4. Synthesis of the ligandsAs part of this work, several polyphosphate ligands were synthesized. The li-gands, EDTMP, DTPMP, TTHMP, DOTMP and CTMP were labeled with 177Lu and investigated (Fig. 9.4). Syntheses of all the ligands were carried out using Mannich-type reaction in strong acidic medium [109]. Ethylene diamine, di-ethylene triamine, triethylene tetramine, cyclen and cyclam were used as start-ing materials for the synthetic backbone of the ligands, EDTMP, DTPMP, TTHMP, DOTMP and CTMP, respectively. Briefl y, the synthesis was carried out as follows. Stoichiometric amounts of the respective amines and anhydrous orthophosphorous acid were dissolved in 37% hydrochloric acid and the result-ing solutions were refl uxed. Appropriate amounts of formaldehyde were added to the refl uxing solution and the refl uxing was continued for another 1 h. The reaction mixture was cooled to room temperature and poured into absolute ethanol with vigorous stirring. The crude products obtained were recrystallized from aqueous ethanol or methanol. The ligands were characterized by IR and 1H-NMR. The details of the synthesis and characterization can be accessed in the published papers [9, 11]. 9.7.1.5. Preparation of 177Lu complexesVarious parameters such as ligand concentration, pH of the reaction mixture and incubation time were needed to be optimized to achieve the best complexa-

203

tion yield for each ligand with 177Lu. Typically, the 177Lu complexes were prepared by dissolving the needed amount of ligand in 0.4 ml of 0.5 M NaHCO3 buffer (pH = 9). To the resulting solutions, 0.1 ml of 177LuCl3 solution (~100 μg of Lu) was added after the addition of 0.5 ml normal saline. The pH of the resulting mixture was adjusted to 9 for EDTMP, TTHMP and CTMP, while it was ad-justed to 7 for DTPMP and DOTMP. The complexation reaction was found to be fast and complete in 15-30 min when incubated at room temperature.

The radiolabeling yields were determined by paper chromatography using ammonia : ethanol : water (1:10:20) and by paper electrophoresis techniques using Whatman 3 MM chromatography paper strips. Paper electrophoresis was

Fig. 9.4. Polyaminophosphonates used as chelating agents for 177Lu [9, 11]

Ligand 1: EDTMP (N,N,N’,N’-tetrakis(phosphonatomethyl)-

ethane-1,2-diamine)

Ligand II: DTPMP (diethylenetriamine pentamethylene phosphonate)

Ligand III: TTHMP (triethylenetetramine hexamethylene phosphonate)

Ligand IV: DOTMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-

methylene phosphonate

Ligand V: CTMP (1,5,8,12-tetraazacyclododecane-1,4,7,10-tetramethylene-phosphonate)

204

carried out for 1 h under a voltage gradient of ~10 V/cm in 0.025 M phosphate buffer (pH 7.5). The results of the complexation studies are summarized in Table 9.6.

Among acyclic ligands better complexation yields were obtained with EDTMP (20:1) where as the other two ligands needed higher molar ratios of ligand to metal. The best complexation yields were obtained with DOTMP which showed 96% complexation yield at 1:1 metal : ligand ratio which in-creased to 99% at 1:2 metal : ligand ratio. The cyclic ligand CTMP was found to be a poor chelating agent for 177Lu with only 75% complexation yield even with 40 mg of the ligand. This was as expected due to the low ionic size of the Lu+3 ion. All the complexes were found to be stable for at least 14 days. 9.7.1.6. Bioevaluation studies All the biodistribution studies were carried out in compliance with the na-tional laws pertaining to the conduct of animal experiments. Bioevaluation of the 177Lu complexes were carried out in Wistar rats weighing 200 ± 300 g by injecting ~200 μl of the complexes, containing 0.5 mCi (15-20 MBq) of the activity, through the tail vein. The animals were sacrifi ced by cardiac puncture post-anaesthesia, at the end of 3 h, 24 h, 48 h and 96 h post injection. Three rats were used for each time point. The tissues and the organs were excised and activity measured in a fl at type NaI(Tl) scintillation counter.

The summary of the results of the biodistribution studies of the four 177Lu labeled phosphonate ligands at 3 h and 24 h are given in Table 9.7. All the complexes showed high bone uptake within 3 h post injection. The observed uptake in tibia was found to be 6.51 ± 0.15, 6.16 ± 0.74, 6.58 ± 0.14, 5.23 ± 0.77%ID/g at 3 h and 7.71 ± 0.70, 6.25 ± 0.65, 7.28 ± 0.30, 5.50 ± 0.59%ID/g

Table 9.6. Complexation yields of phopshonate ligands with 177Lu at optimized pH

Metal : ligand ratio

177Lu-EDTMP 177Lu-DTPMP 177Lu-TTHMP 177Lu-DOTMP% complexation yields

1:1 n.d. n.d. n.d. 95.71:2 n.d. n.d. n.d. 98.91:4 n.d. n.d. n.d. 99.11:5 98.6 98.1 n.d. n.d.1:8 n.d. n.d. n.d. 99.01:10 99.2 98.3 96.4 n.d.1:15 99.2 98.8 96.8 n.d.1:16 n.d. n.d. n.d. 99.51:30 99.4 99.1 97.1 n.d.1:60 n.d. n.d. 99.7 n.d.

n.d. – not done

205

at 24 h post injection for 177Lu-EDTMP, 177Lu-DTPMP, 177Lu-TTHMP and 177Lu-DOTMP, respectively. The highest uptake and retention was observed with 177Lu-EDTMP, followed by 177Lu-TTHMP. Less uptake was seen for 177Lu-DOTMP. The blood pool activity was cleared relatively well for all the complexes and the best blood clearance was seen for 177Lu-EDTMP and 177Lu-DOTMP. No signifi cant uptake was observed in any of the major organs except kidneys. However, the level of uptake was as expected for an agent that will be cleared through the renal system. The liver uptake was low for all the complexes with the maximum uptake being observed for 177Lu-DTPMP, which was probably due to slightly poor complexation yield with this ligand. An important conclu-sion was that none of the complexes showed leaching of the bone activity at least up to 96 h [9, 11]. Considering the long half-life of 177Lu the non-leacha-bility from bone is an important characteristic. In the event of the leaching of the activity from the bone, it would result in redistribution of activity into other organs and will not deliver the desired radiation dose to the bone.

Major conclusions were drawn from the preliminary screening studies with the phosphonate ligands. Among the acyclic complexes, 177Lu-EDTMP and 177Lu-TTHMP were found to be equally suitable with respect to the biodistribu-tion characteristics. However, for getting the maximum complexation yields, 5 mg of EDTMP was suffi cient whereas 25 mg of TTHMP was needed, which weighed against the latter ligand. EDTMP also had the capability of forming complexes over a wide range of pH as compared to the other two acyclic ligands. Among the cyclic ligands, DOTMP showed very high complexation yields at lower ligand : metal molar ratios. CTMP showed very low complexation yields

Table 9.7. Biodistribution pattern of 177Lu complexes

Organ % Uptake (injected dose/gram)

177Lu-EDTMP 177Lu-DTPMP 177Lu-TTHMP 177Lu-DOTMPTime 3h 24 3h 24 3h 24 3h 24hTibia 6.51

(0.15)7.71

(0.70)6.16

(0.74)6.25

(0.65)6.58

(1.14)7.28

(0.30)5.23

(0.77)5.50

(0.59)Blood < 0.01 < 0.01 0.04

(0.04)0.00

(0.00)0.05

(0.04)0.00

(0.00)< 0.01 < 0.01

Kidney 0.43 0.03

0.23 (0.02)

0.81 (0.46)

0.34 (0.29)

0.48 (0.19)

0.37 (0.01)

0.35 (0.08)

0.26 (0.03)

Liver 0.16 (0.02)

0.12 (0.02)

0.32 (0.17)

0.21 (0.12)

0.15 (0.05)

0.13 (0.01)

0.13 (0.02)

0.07 (0.01)

Intestine 0.02 (0.00)

0.07 (0.03)

0.06 (0.05)

0.19 (0.13)

0.06 (0.05)

0.07 (0.02)

0.43 (0.19)

0.15 (0.02)

Muscles 0.01 (0.00)

0.03 (0.03)

0.00 (0.00)

0.00 (0.00)

0.01 (0.01)

0.01 (0.01)

< 0.01 < 0.01

n.d. – not done

206

and this was as expected as the 12 member ring based on cyclen was more compatible with the ionic size of Lu3+ as compared to the 14 member ring based on cyclam in CTMP. The major advantage of the cyclic ligand was that it showed very high complexation yields with lower ligand : metal ratios. This tempted further detailed evaluation of 177Lu-DOTMP [97].

9.7.2. PHARMACOKINETICS AND TOXICITY STUDIES WITH 177Lu-EDTMP [17, 18]9.7.2.1. Rationale for selection of EDTMP The preliminary screening of the 177Lu-polyphosphonate complexes suggested that EDTMP and DOTMP have more favorable properties to be taken up for further studies [9, 11]. DOTMP had the advantage that complexation can be done with lower amounts of ligand whereas EDTMP complex showed higher bone uptake. Ideally both EDTMP and DOTMP could be selected for clinical trials; however, EDTMP was selected as the lead molecule for the development of the radiopharmaceutical. The reasons are as follows:

• 153Sm-EDTMP is already an approved radiopharmaceutical for the same indication and large volumes of data are available with its clinical use in several thousands of patients. Toxicological data for the ligand EDTMP is already established.• Regulators, nuclear medicine physicians and oncologists are familiar with • 153Sm-EDTMP and hence the acceptability of 177Lu-EDTMP is likely to be better than 177Lu-DOTMP. • 177Lu-EDTMP could be considered as an analogue of 153Sm-EDTMP, rather than an altogether new product. This was expected to facilitate a favorable approval procedure for clinical trials.

9.7.2.2. Cold kit formulation A freeze dried kit of EDTMP suitable for 177Lu labeling was envisaged to be a better option rather than the transportation of the radiopharmaceutical from the site of production to the nuclear medicine department. It was also rational to keep the kit composition as close to the originally approved formulation of Quadramet® in terms of ligand quantity Ca+ and Na+ cation contents in order to get an easy approval from the ethics committees. The formulation of the kit was done by Polatom Centre, Poland under the guidance of Dr. Renata Mikołajczak.

The kit consisted of 35 mg EDTMP, (0.077 mM), 5.72 mg CaO, (0.102 mM), 14.1 mg NaOH (0.35 mM) in freeze-dried form. The kit is to be formu-lated by the addition of 1 ml of water of injection, 177LuCl3 (up to 150 mCi) and fi nally adjusting the volume to 5 ml by the addition of normal saline. The reconstituted kit was incubated for 30 min at room temperature prior to adminis-tration of the activity in patients. The radiochemical purity of 177Lu-EDTMP is determined by ITLC (using water : methanol : NH4OH (20:20:1 v/v) as solvent system) in which the 177Lu-EDTMP moves to the solvent front while the un-complexed 177Lu remained at the point of spotting. 177Lu-EDTMP complex

207

having > 99% radiochemical purity was usually obtained which is used for animal or human experiments. 9.7.2.3. Biodistribution studies The biodistribution, pharmacokinetic analysis and imaging studies were per-formed at the National ‘Frédéric Joliot-Curie’ Institute of Radiobiology and Radiohygiene, Budapest, Hungary under the supervision of Dr. Gyozo Janoki. Necessary clearance from the relevant ethical committees was obtained prior to the experiments. Biodistribution studies in mice

177Lu-EDTMP, 8-50 MBq, activity used depending on the duration of animal experiment, was injected in mice (fi ve animals for each time point) intravenously via the tail vein. Mice were sacrifi ced at different time points start-ing from 30 min and ending at 8 week post injection of the tracer. At the ap-propriate time points, mice were euthanized with intra-peritoneal injection of a veterinary euthanasia agent, T-61® ad us. vet. (Bayer Inc., Leverkusen, Germany). Organs of interest, femur bone, bone marrow, muscle, heart, lung, liver, kidneys and thyroid glands were dissected, washed, dried and weighed. About 0.2-0.5 ml of blood was collected by cardiac puncture to measure the blood activity. Bone marrow samples were obtained by rinsing a random femur head, lumen and condyli with 0.5 ml of physiological saline and collecting the washings together with the bone marrow mass. Radioactivity was measured in a well-type gamma counter previously calibrated for 177Lu. Uptake of injected activity in whole organs were calculated and expressed as percent of injected activity (%IA) per organ/tissue.

Pharmacokinetic parameters were derived from the mean values of biodis-tribution data acquired at different time points by applying Topfi t v2.0 phar-macokinetic software (Fischer Verlag, Jena, Germany) using the non-compart-mental model. Time-activity curves were drawn using Prism v5.0 software (GraphPad Inc., USA). The results of the biodistribution studies in mice at different time points studied showed fast accumulation of activity in the bone peaking at 1 h post injection (40.82 ± 3.98%IA in bone corresponding to 19.22 ± 1.81%IA per gram of bone). Activity accumulation was primarily observed in the bones with fast excretion via the renal route. No organs other than bone showed retention of activity in signifi cant amount at any time point. SPECT-CT studies in mice

CD-1 mice were injected with 50 μl (30 MBq) of 177Lu-EDTMP and 3 h post injection, mice were anaesthetized by intraperitoneal (i.p.) injection of ketamine (4 mg/kg body weight) (SBH-Ketamin, SBH Ltd.) and xylazine (1 mg/kg bw) (Xylavet, Alfasan, Holland). A dedicated small animal imaging system, NanoSPECT/CT® (Mediso Ltd., Budapest, Hungary) equipped with a multiplexed multi-pinhole (9 pinholes, aperture 1.4 mm) collimator was used to acquire whole-body SPECT/CT images. Energy window was centered at 208 keV with ± 10% width for all imaging studies. Acquisition times were defi ned

208

to obtain 100 000 counts for each projection with 24 projections. Images and maximum intensity projections (MIPs) were reconstructed using the dedicated softwares Invivoscope (Bioscan Inc., Washington, USA) and Mediso Inter-ViewXP™ (Mediso Ltd., Budapest, Hungary). Figure 9.5 shows maximal in-tensity projection SPECT images in a mouse overlaid on CT images. Autoradiography in mice

Whole body autoradiography studies were done in mice injected with 5 MBq of 177Lu-EDTMP. The animals were euthanized at 3 day post injection and frozen in liquid nitrogen. 50 μm thin cryostat sections were prepared using Leica CM 3600 instrument (Leica Co., Germany). The sections together with activity standards containing 0.18 kBq, 1.8 kBq and 9.2 kBq were exposed to phosphor imaging plates (Molecular Dynamics Phosphor Imager, GE Health-care, USA). Results of the digital autoradiography study in mouse are presented in Fig. 9.6 which clearly illustrates the distribution of 177Lu-EDTMP with pre-dominant accumulation in the skeleton. Internal reference activity sources are also shown in the fi gure (black round spot). Autoradiography studies showed up-take of the activity by both long and spongeous bones in the remodeling regions and bone surfaces. Joints and surfaces of vertebrae and the ischiadic bone ac-cumulated more radioactivity than the internal spongiosa of the same bones. Imaging studies in rats

About 40 MBq of 177Lu-EDTMP was injected into the tail vein of 3 rats and imaged after 3 h using the NanoSPECT/CT system as described above. The anesthesia and imaging parameters used were same as described above except that a pinhole aperture with 2.0 mm diameter was applied. Figure 9.7 shows the SPECT/CT images of a rat and the distribution of the radiopharma-ceuticals was similar to the mice images.

Figure 9.5. Maximal intensity projection rendering and fusion of mouse CT and SPECT with 177Lu-EDTMP. Projection image top shows vertebral column and bottom shows right knee joint.

209

Fig.9.6. Digital autoradiography of saggital sections of a mouse at 3 days post injec-tion 177Lu-EDTMP. Spots at the right side of the image represent standards of 0.18 kBq, 1.8 kBq and 9.2 kBq of 177Lu in order of brightness, respectively

Fig. 9.7. SPECT/CT images of Wistar rats at 3 h post injection with 40 MBq of 177Lu-EDTMP. Projection image top shows midline and bottom shows right knee joint [18]

210

Biodistribution and imaging studies in rabbitsNew Zealand White rabbits, three animals for each time point were taken

and the studies were done from 30 min to 8 weeks. 177Lu-EDTMP tracer was injected via the auricular vein. Rabbits used for early time points (30 min to 1 week) received 100 MBq (in 0.3-0.4 ml) while the ones used for later time point studies received 200 MBq (in 0.6-0.7 ml) of activity. Animals were anaesthetized with intramuscular injection of ketamin (dose 100 mg/kg bw; bw – body weight) and xylazine (dose 10 mg/kg bw). One ml of blood was drawn from the heart and then the animals were euthanized with intra-cardial injection of 1 ml T-61® ad us. vet. bone (tibia was taken as sample), bone marrow, muscle, heart, lung, liver, kidneys and thyroid glands were collected. Organs were cleaned, dried and weighed. Radioactivity in the organ was measured in a well-type gamma counter. Bone marrow was obtained by physical removal of it from the tibial plateau, epicondyli and from the tibial lumen. In rabbits, percent organ--contribution to body weight were assumed as bone mass 5%, blood 8%, bone marrow 8% and muscle 50% of body mass.

The results of the biodistribution studies in rabbits were comparable to those obtained in mice. At 3 h 36.91 ± 8.92%IA of the injected activity is ac-cumulated in bone with no other organ having activity retention higher than 1%IA. No measurable activity was seen in any other organ than bone after

Fig. 9.8. Scintigraphic images of a rabbit 1 h (A), 1 day (B), 1 week (C) and 4 weeks (D) post injection of 40 MBq of 177Lu-EDTMP

A

C

B

D

211

48 h. The results of the biodistribution studies were used to calculate the phar-macokinetics parameters.

Ventrodorsal images of rabbits at different time points (1 h, 1 d, 1 week and 4 weeks) post injection are presented in Fig. 9.8 which shows the accumulation of the radiopharmaceutical in the bone. At 1 h post injection activity in bladder is observed. The images at later time points show activity in no other organ other than the bone. Imaging studies in dogs

Imaging studies in dogs consisted of ventrodorsal (VD) and dorsoventral (DV) views by using a Nucline XRing/R (Mediso Ltd., Hungary) gamma camera equipped with a high resolution general purpose (HRGP) collimator. Whole-body (512x1024) and spot images (256x256) were acquired at different time points starting at 30 min and up to 4 weeks post injection. Prior to imag-ing, animals were anaesthetized by i.v. injection of ketamin and xylazine at a dose of 100 mg/kg and 5 mg/kg bw, respectively. Images were processed with the Pmod 2.7 software package (Pmod Technologies Co., Zürich, Switzerland).

Fig. 9.9. Scintigraphic images of a dog injected with 9.25 MBq/kg bw 1 h (A) and 1 week (B) post injection, and another dog injected with 37 MBq/kg bw 1 h (C) and 1 week (D) post injection of 177Lu-EDTMP

A B

C D

212

The ventrodorsal images of two dogs at 1 h and 1 week post injection of 177Lu-EDTMP are given in Fig. 9.9. Upper panel (A and B) represents the dog which received 9.25 MBq/kg bw and the lower panel (C and D) is the dog which received 37 MBq/kg bw. Accumulation of activity in bladder is seen in the bone and bladder at 1 h time point. At day one activity was seen only in the bone. There was no change in the images up to 4 weeks studied clearly indicat-ing that the 177Lu-EDTMP once accumulated to the bone is not released, a property needed for the bone seeking radiopharmaceuticals.

Imaging studies in mice, rats, rabbits and dogs showed rapid bone uptake together with fast blood clearance of the tracer. Prolonged bone localization was seen in all species studied with no leakage of bone accumulated activity into any of the organs.

There was no difference in the uptake or excretory pattern among the four different activity groups of dogs used for dose escalation studies (9.25, 18.5, 27.75 and 37 MBq/kg bw). The non-bone accumulated activity was excreted preferentially through the renal route. By pooling the results of the animals of all groups, it was inferred that > 96% of the urinary excretion occurs in the fi rst 24 h. The results provided strong support to the inference that there will not be any signifi cant radiation related adverse effects to any organs other than bone due to activity accumulation. Activity uptake in bone marrow was also insignifi cant. 9.7.2.4. Pharmacokinetic studies Pharmacokinetic parameters in mice and rabbits were calculated from the mean values of biodistribution data acquired at different time points by applying Topfi t v2.0 pharmacokinetic software (Fischer Verlag, Jena, Germany) using the non-compartmental model. Time-activity curves were drawn using Prism v5.0 software (GraphPad Inc., USA). Pharmacokinetic parameters in mice and rabbits gave important information about the biokinetics of 177Lu-EDTMP. Detailed analysis can be seen in the published paper [18]. Results of the pharmacoki-netic modeling studies support stable and long lasting binding of 177Lu-EDTMP to bone. The terminal half-life of 177Lu-EDTMP in bone was 2130 h and 1870 h where as in blood was 7.3 h and 8.9 h in mice and rabbits, respectively. The results suggested near identical pharmacokinetic characteristics in the two species. All organs other than bone having long terminal elimination half-lives contained minuscule fraction of radioactivity as refl ected by very small values of area under the curve (AUC∞). The bone-to-muscle ratio of AUC∞ was 130 while the bone-to-blood ratio was > 14 000 in mice. In rabbits, the AUC∞ ratios were 732 for bone-to-muscle and 2811 for bone-to-blood. 9.7.2.5. Toxicity studies in healthy Beagle dogsDose escalation studies with four activity levels (9.25, 18.5, 27.75 and 37 MBq/kg) were done to study the toxicity of 177Lu-EDTMP. Three randomized healthy male beagle dogs weighing between 13 kg and 23 kg were used for each activity level. The tracer was injected through the cephalic vein. Venous blood samples (4 ml, cephalic or jugular vein) were drawn from each of the

213

injected animals to determine the biochemical parameters. Serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALKP), gamma-glutamyl transferase (GGT), urea and creatinine were measured. Urea/creatinine ratio was calculated to verify possible renal damage. Automated complete blood counts (CBC) performed included white blood cells (WBC), red blood cells (RBC) and platelets (PLT). All measurements were done by a dedicated veterinary laboratory (VetMedLabor Ltd., Budapest, Hungary). Toxicity to bone marrow was defi ned as the reduction in platelet concentration below 100 g/l (103/μl) or WBC level below 4 g/l.

Cumulative urine up to 72 h was collected in fractions during the time intervals 0-6 h, 6-24 h, 1-2 days and 2-3 days via an indwelling Foley catheter. At each time point the urinary bladder was rinsed with saline and the fi nal volume was documented. Activity in the corresponding pooled fractions was

Fig. 9.10. White blood cell (A) count and platelet count (B) of the means of 3 animals in each dose group over time

A

B

214

determined in 1 ml samples using the NZ-310 gamma counter. Activity ex-creted in the urine during different time intervals of the four different activity groups of dogs was estimated. The results suggested that most of the non-bone accumulated activity was excreted between 0-6 h. Activity excretion during 48-72 h was insignifi cant suggesting no leakage of the activity from the bone.

All the biochemical parameters studied are within the normal range. Serial measurements of white blood cells and platelets for all the activity groups are presented in Fig. 9.10. The normal range for white blood cells and the lower cut of limit for platelets are shown with horizontal bars. Neither WBC nor platelet counts reduced below toxic levels in any of the activity group studied. Reduction of platelet counts below the normal range was seen at 1 week and persisted till 4th week in the case of dogs administered with the highest activ-ity. The counts increased to normal range 5 week post administration of activ-ity. The follow-up on the dogs by the veterinary physician did not report any changes or deterioration on the overall health status of the animals. In three dogs mild, transient urethritis was observed which was attributed to catheteri-zation.

Drop below normal values for platelet and WBC counts was seen in the highest activity group. As opposed to platelets, the trend in the WBC counts was not unidirectional. This could be probably due to a reactive leukocytosis to urethritis caused by catheterization. This change in WBC counts indicates that the proliferative capability of the bone marrow in dogs is not hindered even at the highest level of injected activity. Neither WBC nor platelet counts reached the toxicity levels.

Biochemical parameters were measured to assess the liver and kidney function which also did not reveal any abnormalities for all the four activity levels studied. The tracer being excreted mainly through the renal pathway, the kidney and bladder are the critical organs. Measured biochemical parameters as well as serum urea to creatinine ratio did not indicate any damage to the renal system, even with the highest level of injected activity. The results of the dose escalation studies clearly suggested that there are no adverse effects or toxicity up to 37 MBq/kg bw of injected activity.

The biodistribution, pharmacokinetic, biochemical and haematologic para-meters of 177Lu-EDTMP in different species were done in order to collect data needed to support a human clinical trial. The studies of 177Lu-EDTMP clearly demonstrated that the tracer is taken up almost exclusively by the skeletal system, with minimal activity accumulation in any other organ. Multidose studies up to 37 MBq/kg bw in dogs did not result in any toxicity as seen from the biochemical parameters and hematological measurements. Based on the results of the present studies, 177Lu-EDTMP was considered as a suitable radio-pharmaceutical to be taken up for human clinical trials. Data collected from the studies formed the basis for initiating a multicenter clinical trial of 177Lu-EDTMP under an IAEA Coordinated Research Project (CRP).

215

As part of the clinical trial, the radiopharmaceutical, 177Lu-EDTMP, was taken up for a pre-Phase I clinical trial at the All India Institute of Medical Sciences in order to generate human pharmacokinetic and dosimetry informa-tion. For this purpose tracer dose of 5 mCi (185 MBq) was injected in fi ve patients suffering from prostate cancer. The uptake of tracer in patients is shown in Fig. 9.11. One of the patients had exhaustive bone metastasis. The radiopharmaceu-tical is currently undergoing a multi-country–multicenter Phase I/II clinical

trial under an IAEA CRP under the responsibility of Dr. J.J. Zaknun of the Nu-clear Medicine Section. Upon completion of this IAEA CRP, it is expected that suffi cient data will be collected which will enable the approval of 177Lu-EDTMP as a radiopharmaceutical for bone pain palliation in the participating countries.

9.7.3. 177Lu-DOTA-LANREOTIDE [14] Virgolini et al. reported the use of 111In labeled lanreotide, β-Naphthyl-Ala--Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2, a disulfi de linked cyclic octapeptide and a somatostatin analogue for imaging of a wide variety of tumors expressing somatostatin receptors [110]. The targeting of neuroendocrine tumors, intestinal adenocarcinomas and lymphomas with 111In labeled lanreotide for diagnostic purposes prompted IAEA to take up lanreotide as a lead molecule in one of the Coordinated Research Projects in which the author participated as the chief scientifi c investigator from India. The development of 90Y- and 125I labeled lanreotide was carried out as part of the CRP work [14].

As part of the above project DOTA-lanreotide conjugate was already pre-pared for labeling with 90Y. Hence, it was only logical to study radiolabeling of lanreotide with 177Lu. The synthesis of DOTA-lanreotide, radiolabeling with 177Lu and its bioevaluation studies are described. Lanreotide with a free NH2

Fig. 9.11. SPECT images with 177Lu-EDTMP in a patient with prostate cancer with (left: two images, anterior and posterior) and without metastasis (right: two images, anterior and posterior) (Courtesy C.S. Bal, AIIMS, India)

216

residue at the N-terminal position provided a facile access to designing a lan-reotide-DOTA conjugate (Fig. 9.12). 9.7.3.1. Synthesis of DOTA-LAN conjugateThe synthesis of DOTA-LAN conjugate was carried out via a three-step syn-thetic procedure as depicted in Fig. 9.13 starting with lanreotide obtained from PiChem, Austria.

Step 1: The protection of the free amino residue of lysine in lanreotide (LAN) was carried out with ditert-butyl dicarbonate (BOC-anhydride) to yield BOC-protected lanreotide (LAN-BOC) (I).

Step 2: The LAN-BOC-DOTA (II) conjugate was formed at the β-naphthyl-amino residue of alanine. The peptide bond formation was effected via the for-mation of the N-hydroxy-succinimide ester of DOTA in the presence of dicyclo-hexyl carbodiimide (DCC) as the condensing agent. LAN-BOC obtained in step 1 was dissolved in (1:1) double distilled water : dimethyl formamide and to the resulting solution, N-hydroxy-succinimide, DCC and DOTA were added. The pH of the solution was maintained at ~9 by the addition of 1 M NaOH solution. The reaction mixture was kept stirring for 16 h at room temperature following which turbidity was observed. The solvent was removed under a slow stream of nitrogen to obtain the compound II.

Step 3: DOTA-LAN: De-blocking of the BOC-protected NH2 group was done to yield the conjugated product DOTA-LAN. The deprotection was carried out by stirring compound II in dichloromethane (1.5 ml) with trifl uoroacetic acid (TFA) at room temperature for 30 min. Then the solvent was removed under nitrogen whereby the crude conjugate which was obtained. This crude product obtained was purifi ed by preparative TLC technique. Purifi ed DOTA-LAN

Fig. 9.12. Pictorial representation of DOTA-lanreotide

H

ONH

NH

ONH

SH

O OH

HN

H

H

O

HN

NH2

HNO

H

H

O

NHH3C

CH3

H

H

S

O

HN

HCONH2

H

OHH3C

(Ala)

(Cys)(Cys)

(Tyr)

(Trp)(Lys)

(Val)

(Thr)

LANREOTIDE

NN

NN

COOH

COOH

HOOC

C

O

α

β

N

-DOTA

217

was characterized by a combination of several spectroscopic techniques such as, UV, FT-IR and 1H-NMR spectroscopy. 9.7.3.2. Radiolabeling of DOTA-LAN conjugate with 177LuThe radiolabeling of DOTA-LAN conjugate was done by adding 20 μl 177LuCl3 (20 MBq, 0.002 μmole Lu) solution to a 200 μl solution of DOTA-LAN con-jugate (0.1 μmole) in 0.5 M sodium bicarbonate buffer at pH 9. The reaction

Fig. 9.13. Synthetic scheme for the preparation of DOTA-LAN

218

mixture was incubated at 37οC for 2 h after adjusting the pH to ~5 with 0.1 M HCl. The complexation of the DOTA-LAN conjugate was also studied in 0.1 M sodium acetate buffer, pH 5.5 by similar manner. The estimation of the radio-chemical purity and fi nal purifi cation of the labeled product was carried out by HPLC analysis using gradient elution technique using a C-18 reversed phase column. Water (A) and acetonitrile (B) mixtures with 0.1% trifl uoroacetic acid were used as the mobile phase. The HPLC chromatogram of 177Lu-DOTA-LAN conjugate is shown in Fig. 9.14. Radiochemical purity as estimated by HPLC was found to be ~85% under optimum radiolabeling conditions. The radiola-

beled conjugate was purifi ed by HPLC. The stability of the radiolabeled con-jugate was studied by paper chromatography and HPLC, and the product was found to be stable up to 48 h at room temperature with > 80% of its radio-chemical purity retained. 9.7.3.3. In vitro cell binding studiesFor achieving therapeutic effect of targeted radiopharmaceuticals such as radio-labeled somatostatin analogues, the radiochemical preparation should be re-tained within the cell after binding and hence cell binding studies are evolving as crucial techniques for the in vitro evaluation of therapeutic radiopharmaceu-ticals. Three cell lines, A-431 human epidermoid carcinoma, IMR-32 human brain neuroblastoma and DU-145 human prostate carcinoma cell lines were used for the in vitro cell binding studies. Of the three different cell lines studied, A-431 and IMR-32 cells showed signifi cant uptake of the tracer when incu-bated with 177Lu-DOTA-LAN corresponding to peptide concentration below 75 nM. There was no uptake observed with DU-145 cells at the same peptide concentration. In case of A-431 cells, at 71 nM radiotracer concentration, when inhibition was carried out with cold lanreotide, the binding dropped from 17%

Fig. 9.14. HPLC pattern of 177Lu-DOTA-LAN conjugate using a gradient elution of acetonitrile (A) and water

0 200 400 600 800 1000 1200 1400 1600 1800

0

50

100

150

200

250

300

Uncomplexed 177Lu

177Lu-DOTA-Lanreotide

Cou

nts/

sec

Retention time

219

to 0.6%, indicating the specifi city of the prepared radiochemical preparation for somatostatin receptors.

In the studies with 177Lu-DOTA-LAN, approximately 35% of the cell as-sociated activity remained internalized after removal of surface bound activity with 20 mM aqueous sodium acetate solution at 30 min post-incubation. Pre-liminary in vitro cell binding studies using 177Lu-DOTA-LAN tracer offered considerable promise towards its potential use as an agent for targeted tumor therapy.

9.7.4. 177Lu LABELED DOTA-ESTRADIOL [16]Development of potential radiotherapeutic agents based on 177Lu for targeting tumors overexpressing steroidal receptors is important and hence the develop-ment of 177Lu labeled estradiol was taken up. Our interest in this work was due to the expertise in the development of radioimmunoassay for steroid hormones which gave experience in the synthesis of several derivatives of different steroid hormones both for radiolabeling with 125I as well as for the preparation of anti-bodies.

With 17β-estradiol as the available precursor, derivatization of the C6 posi-tion appeared to be more feasible, as compared to the C7 and C11 positions, in an attempt to prepare a potential agent for radiotherapy. Besides being distant from the C3 and C17 positions, the C6 position in the estradiol molecule have the additional advantage of being the benzylic position with respect to the aro-matic ring and therefore becomes a suitable site for modifi cation and attachment of a BFCA. The attachment of a DOTA derivative required the relatively less sterically hindered viz. the α disposition of a suitable functionality at the C6 position. It was therefore decided to investigate the feasibility of covalently coupling the 6α-amino-estradiol with a suitable DOTA derivative. Since, para-

-thiocyanato-benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-NCS-benzyl-DOTA), the thiosphosgene activated synthon from the precur-sor para-amino-benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-NH2-benzyl-DOTA) was readily available, the covalent coupling with the amino substituent was quite feasible. Hence, this bioconjugation route was selected to synthesize DOTA-estradiol (Fig. 9.15).

Fig. 9.15. DOTA-estradiol

S

C

N

N

N

N

CH2COOHHOOCH2C

HOOCH2C CH2COOH

NH

OH

HO

NH

220

9.7.4.1. Synthesis of p-NCS-benzyl-DOTA-estradiol conjugateThe synthesis of 6α-amino-17β-estradiol (IV) was achieved by a fi ve-step synthetic procedure starting with (Fig. 9.16). The fi rst step involved the protec-tion of the two hydroxyl groups of 17β-estradiol by acetylation. In step 2, corresponding keto derivative was synthesized by oxidation with chromic oxide. Step 3 involved conversion of the 6-keto derivative to 6-amino deriva-tive by reaction with sodium cyano borohydride followed by the deprotection

of the hydroxyl groups by base hydrolysis. The fi nal step involved the conjuga-tion of 6α-amino-17β-estradiol with isothiocyanato benzyl DOTA to get the target molecule. The characterization of all the intermediates as well as the fi nal product was achieved by spectroscopic techniques, such as, FT-IR and 1H-NMR spectroscopy and the details can be accessed from the published paper [16]. 9.7.4.2. Radiolabeling of p-NCS-benzyl-DOTA-estradiol with 177LuReaction parameters were varied in order to determine the optimum conditions to obtain maximum complexation yield of 177Lu-p-NCS-benzyl-DOTA-estra-diol complex. The optimized conditions were as follows: 300 μg of the ligand was dissolved in 0.2 ml of dimethyl formamide, followed by the addition of 0.2 ml of 0.1 M ammonium acetate buffer (pH ~5.5) and 40 μl 177LuCl3 solution (100-200 MBq). The volume of the reaction mixture was made up to 1 ml us-

Fig. 9.16. Synthesis of p-NCS-benzyl-DOTA-6α-amino-17β-estradiol conjugate

221

ing normal saline and its pH was adjusted to ~5. Finally the reaction mixture was incubated at 37οC for 2 h.

The characterization of 177Lu-p-NCS-benzyl-DOTA-estradiol complex was carried out by a combination of paper chromatography and paper electro-phoresis techniques. Purifi cation of the 177Lu labeled p-NCS-benzyl-DOTA-es-tradiol conjugate was achieved by using Sep-pak® column as well as by high performance liquid chromatography (HPLC) technique. HPLC studies also helped in the precise determination of the extent of complexation. The stability of the 177Lu labeled p-NCS-benzyl-DOTA-estradiol conjugate was studied by using the similar quality control techniques and was observed that the complex exhibited good stability when stored at room temperature as it maintained its radiochemical purity to the extent of 77% for a period of 7 days after prepara-tion.9.7.4.3. Antibody binding studiesThe 177Lu labeled p-NCS-benzyl-DOTA-estradiol conjugate was incubated with antibodies raised against estradiol-6-carboxymethyloxime-bovine serum albu-min (BSA) conjugate. The antibody used for the present study was validated in a separate radioimmunoassay system for serum estradiol estimation. The binding of the reaction mixture as well as the purifi ed fractions of the tracer was studied.

About 0.1 ml of the diluted complex (~1 μg) was incubated with 1:5 di-luted antiserum in 0.05 M phosphate buffer containing 0.1% BSA. About 0.1 ml of 2% γ-globulin was added in all the tubes to facilitate precipitation. The incubation was carried out for 2 h at room temperature after which, 1 ml of 22% polyethylene glycol in saline was added to precipitate the 177Lu-p-NCS--benzyl-DOTA-estradiol complex bound to antibody. The precipitate was separated by centrifugation and the radioactivity in the precipitate was meas-ured. Blank studies were simultaneously set up without the antiserum to rule out any non-specifi c binding.

Binding studies of a 1:5 diluted antibody with the unpurifi ed reaction mix-ture showed that ~9% of the tracer was bound to the antibody. The extent of binding increased to 14% with Sep-pak® purifi ed product. A further improve-ment to a maximum of 38% was achieved on HPLC purifi cation with 300 ng of the 177Lu-p-NCS-benzyl-DOTA-estradiol complex (125 Bq/ng). This com-pared well with earlier studies with radioiodinated estradiol.

The binding observed with antibody indicated that there is no major change in the molecule with respect to antibody recognition after incorporation of the BFCA. The specifi c binding is further confi rmed by the low non-specifi c bind-ing of < 1% observed when 177Lu-p-NCS-benzyl-DOTA complex was reacted with the antibody.9.7.4.4. Cell binding studies The MCF-7, human breast cancer cell line known to possess estrogen receptors, was used for cell uptake studies of the radiolabeled conjugate. The cell binding

222

study protocol was similar to the one used for 177Lu-DOTA-LAN described in the previous section. Cell binding of ~13% was observed with 5×104 MCF-7 cells for 1 μg of the 177Lu-p-NCS-benzyl-DOTA-estradiol complex. On Sep-pak® purifi cation, the cell uptake improved to 17%. No further increase in cell uptake was observed even after using HPLC purifi ed complex. It was observed that the tracer uptake in the cells decreased to ~8.3% on the addition of 100 μg of cold estradiol. The decrease in cell uptake on the addition of estradiol indi-cated the specifi city of the radiolabeled conjugate for the MCF-7 cell lines. Blank experiments were also carried out with 177Lu labeled BFCA under similar experimental conditions. No retention of the activity in the cell pellet was observed, ruling out the possibility of carrier-mediated uptake.

The radiolabeling of the estradiol-BFCA conjugate with 177Lu was the fi rst report on the preparation of a 177Lu labeled steroid hormone and hence a very signifi cant development. There is scope to develop either the presently studied derivative or other suitable steroid derivatives labeled with 177Lu as radiophar-maceuticals with steroid as the vehicle to carry the radioactivity to the tumor site. The fi rst logistical step will be to prepare 68Ga labeled tracers for studying the uptake of the derivatives by the tumor. Based on the tumor uptake of such diagnostic tracers, 177Lu products can be developed for therapy of breast cancer. The product might fi nd good application for the treatment of hormone receptor positive disease post surgery to destroy the remnant tumor.

9.8. CONCLUSIONS

Lutetium-177 has already proven its utility as a key radionuclide for targeted therapy. The major advantage of this radionuclide is the ability to prepare it in large quantities in several countries by ‘direct’ neutron activation of highly enriched target which is commercially available. The specifi c activity of the resultant product is high enough to radiolabel most of the targeting molecules including antibodies and peptides. The ‘indirect route’ of production of the radionuclide will result in substantially higher cost of production without sig-nifi cant advantage with respect to specifi c activity. The small amount of 177mLu present in the ‘direct’ route of production is not a major concern as far as patient safety is concerned.

177Lu-DOTA peptides are already established and the effi cacy of a few of the monoclonal antibody based products is also proven. The clinical trial of 177Lu-EDTMP is expected to help in making it a regular product in routine use for bone pain palliation in many countries. The work on the labeling of estra-diol is very signifi cant as it can open up the possibility of yet another class of small molecule based therapeutic radiopharmaceuticals. There is an ever in-creasing possibility of identifying a wide spectrum of targeting peptides, monoclonal antibodies and other cancer seeking small molecule for the develop-

223

ment of 177Lu based therapeutic radiopharmaceuticals for targeted therapy. The development and application of 177Lu radiopharmaceuticals are expected to grow much faster in the coming years. As the author used to tell to his young colleagues the ‘177Lu is a gold mine for radiopharmaceuticals development’ and the exploration has started only in the top layer of the mine. There is scope to go deeper to realize the full potential of this radionuclide as a therapeutic radio-nuclide.

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