Biodistribution and post-therapy dosimetric analysis of [177Lu]Lu-DOTAZOL in patients with osteoblastic metastases: first results

Background Preclinical biodistribution and dosimetric analysis of [177Lu]Lu-DOTAZOL suggest the bisphosphonate zoledronate as a promising new radiopharmaceutical for therapy of bone metastases. We evaluated biodistribution and normal organ absorbed doses resulting from therapeutic doses of [177Lu]Lu-DOTAZOL in patients with metastatic skeletal disease. Method Four patients with metastatic skeletal disease (age range, 64–83 years) secondary to metastatic castration-resistant prostate carcinoma or bronchial carcinoma were treated with a mean dose of 5968 ± 64 MBq (161.3 mCi) of [177Lu]Lu-DOTAZOL. Biodistribution was assessed with serial planar whole body scintigraphy at 20 min and 3, 24, and 167 h post injection (p.i.) and blood samples at 20 min and 3, 8, 24, and 167 h p.i. Percent of injected activity in the blood, kidneys, urinary bladder, skeleton, and whole body was determined. Bone marrow self-dose was determined by an indirect blood-based method. Urinary bladder wall residence time was calculated using Cloutier’s dynamic urinary bladder model with a 4-h voiding interval. OLINDA/EXM version 2.0 (Hermes Medical Solutions, Stockholm, Sweden) software was used to determine residence times in source organs by applying biexponential curve fitting and to calculate organ absorbed dose. Results Qualitative biodistribution analysis revealed early and high uptake of [177Lu]Lu-DOTAZOL in the kidneys with fast clearance showing minimal activity by 24 h p.i. Activity in the skeleton increased gradually over time. Mean residence times were found to be highest in the skeleton followed by the kidneys. Highest mean organ absorbed dose was 3.33 mSv/MBq for osteogenic cells followed by kidneys (0.490 mSv/MBq), red marrow (0.461 mSv/MBq), and urinary bladder wall (0.322 mSv/MBq). The biodistribution and normal organ absorbed doses of [177Lu]Lu-DOTAZOL are consistent with preclinical data. Conclusion [177Lu]Lu-DOTAZOL shows maximum absorbed doses in bone and low kidney doses, making it a promising agent for radionuclide therapy of bone metastasis. Further studies are warranted to evaluate the efficacy and safety of radionuclide therapy with [177Lu]Lu-DOTAZOL in the clinical setting.


Introduction
Occurrence of painful bone metastases is a frequent complication of solid tumors that reduces the quality of life in many patients [1]. Radionuclide therapy for bone pain palliation in patients with progressive skeletal metastatic disease has been established in the clinical routine already decades ago [2,3]. Several radiopharmaceuticals are or have been in use such as [ 32 [1,2,4]. The therapeutic effect requires the accumulation of these radiopharmaceuticals at osteoblastic sites of metastases and deposition of energy by β − or α particle. These radiopharmaceuticals are used either as monotherapy or in combination with systemic chemotherapy and bisphosphonates [4]. The choice of the radiopharmaceutical is dependent on its inherent properties such as physical half-life, energy and particle range, as well as ease of availability, efficacy, and side effects [5].
Yet, the quest for a stable and effective bone-seeking therapeutic radiopharmaceutical is still an ongoing process. Lutetium-177 with a half-life of 6.73 days, a low range of its β − particles with maximum energy (E βmax = 497 keV), gamma emissions at energies of 112 keV (6.4%) and 208 keV (11%), and the possibility of cost-effective large scale production with high specific activity and radionuclide purity has gained high acceptance as a therapeutic radionuclide [6]. Owing to the deposition of its β − energy in the lesions and their close environment, it is best suited for small-to medium-sized tumor lesions when labeled with a suitable carrier [7]. [ 177 Lu]Lu-DOTA-TOC, [ 177 Lu]Lu-DOTA-TATE [8,9], and [ 177 Lu]Lu-PSMA-617 [10,11] have been proven effective for the treatment of neuroendocrine tumors and metastatic castrationresistant prostate carcinoma (mCRPC), respectively. Moreover, they allow for a good theranostic combination with their gallium-68-labeled imaging counterparts using positron emission tomography (PET) [12].
Zoledronate, nitrogen containing hydroxy bisphosphonate, has recently been conjugated with DOTA (DOTA-ZOL ) and labeled with gallium-68 for imaging and with lutetium-177 for therapy as new theranostic pair for targeting bone metastases [27]. Zoledronate is known to have improved antiresorptive effects owing to its higher hydroxyapatite binding and internalization by osteoclasts with subsequent increased apoptosis. The mode of action is accompanied by inhibition of farnesyl pyrophosphate enzyme of the mevalonate pathway that results in the inhibition of osteoclastic activity [13].

Patient selection
In this retrospective study, we analyzed four patients with metastatic skeletal disease secondary to mCRPC or bronchial carcinoma that were treated between July 2016 and September 2017 with [ 177 Lu]Lu-DOTA ZOL . All treatments were performed in the context of an individual treatment attempt as no other treatment options were left for these patients. Written informed consent was obtained from all patients. The local ethical committee waived the ethical statement due to the retrospective character of the study. All procedures were followed in accordance with ethical standards of the institutional review board and therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and all subsequent revisions.
After confirming sufficient uptake in the bone metastases with [ 68 Ga]Ga-DOTA ZOL PET/CT shown in Fig. 1e, patients were hospitalized in our treatment unit. All patients had normal kidney function confirmed by renal function tests and renal scintigraphy. Patients were administered a mean activity of 5968 MBq (161.3 mCi) (5873-6000 MBq) of [ 177 Lu]Lu-DOTA ZOL intravenously. Table 1 shows patient details along with a history of previous treatments.

Imaging protocol
Serial whole body planar scintigraphy (anterior and posterior views) was performed with dual head Symbia SPECT/CT system (Symbia T, Siemens Healthineers, Erlangen, Germany) at 20 min and 3, 24, and 167 h post injection (p.i). Acquisition was done in the supine position at a speed of 10 cm/min using medium energy parallel hole collimators with 20% energy window centered at a photopeak of 208 keV. Images were processed using an iterative ordered subset maximization algorithm provided by the manufacturer into a matrix of 256 × 1024. The first data set obtained at 20 min (prior to voiding of the bladder) was considered as a reference with 100% of administered activity. A standard source of known activity was placed between the legs in all images at the time of acquisition. For conversion of counts/min to activity, the gamma camera was pre-calibrated using a known activity of [ 177 Lu]Lu-DOTA ZOL and imaging it at the same speed and distance of 10 cm/min.

Blood sampling
One to two milliliters of blood samples was drawn at 20 min and 3, 8, 24, and 167 h p.i. Due to high counts that may lead to errors in measurement, 0.2-ml blood samples were prepared and measured along with 0.2-ml sample from the known standard activity of [ 177 Lu]Lu-DOTA ZOL using a 1480 WIZARD TM 3n Gamma counter. The calibration factor determined from the standard activity measurement was used to determine the activity in blood samples at the respective data points.

Data analysis
The images were qualitatively analyzed to assess the biodistribution of [ 177 Lu]Lu-DOTA ZOL in the whole body and organs. All organs with uptake equal to or more than that of the kidneys were considered as source organs. Kidneys and urinary bladder showed high uptake in the 24-h image data sets and were considered as source organs that included kidneys and urinary bladder. Adductor muscle was measured as soft tissue reference. Whole body ROI's were drawn. A rectangular ROI was drawn near the head region above the shoulder for background measurement and an elliptical ROI was used for measurement of the standard source placed between the legs. Same sized ROI's were replicated on serial images (kidneys ROi's up to the 24-h data set and all remaining ROI's in all subsequent image data sets). Background corrected counts in the right and left kidney, soft tissue, urinary bladder, and whole body were determined on anterior and posterior images. The geometric mean counts/min in all source organs at all data time points was determined. Using EANM dosimetry committee guidelines [31], whole body activity at subsequent time points (T) was determined by multiplying the injected activity with the normalized geometric mean whole body counts at the respective time points as given in Eq. 1.
where t = 20 min, T = subsequent time points, and A 0 = initial injected activity. Likewise, activity in the urinary bladder was also determined by multiplying the injected activity with the normalized geometric mean counts in the urinary bladder.
For calculation of activity in the right and left kidneys at all data points, a conjugate view method with a simple geometrically based subtraction technique given in Eq. 2 [32] was used.
where I A = anterior count rate, I P = posterior count rate, f j represents source organ self-attenuation correction which was calculated from the source region linear attenuation coefficient μ j , and source thickness t j using Eq. 3 [32]. Factor μ e t represents the transmission factor across the patient thickness t in the area of the ROI with a linear attenuation coefficient μ e calculated using Eq. 4 [32]. From [ 68 Ga]Ga-DOTA ZOL -PET/CT of a respective patient, CT-based measurements of source organ thickness as well as whole body thickness and thickness anterior and posterior to source organs at the same level were used. C is the calibration factor determined for gamma camera with a known standard source and was the same in all the studies. For the measurements of μ j and μ i (linear attenuation coefficients for whole thickness), we applied a CT-based Hounsfield unit method described by Kabasakal et al. [33] for [ 177 Lu]Lu-PSMA-617 dosimetric analysis.
A simple geometric based background subtraction technique using Eq. 5 [32] was used.
where I ADJ is the count rate through the patient from a soft tissue area of the same size as that of the organ ROI. I A , I P , t j, and t are the same as previously defined.
Percent injected activity in the whole body, urinary bladder, and kidneys at all time points was determined. To calculate percent injected activity in the skeleton, from percent whole body activity, percent blood, urinary bladder, and kidneys activities were subtracted.

Dosimetric analysis
Percent injected activity in the whole body, kidneys, and skeletal system at all data time points was used to determine residence times (MBq-h/MBq) by fitting biexponential kinetic analysis using OLINDA/EXM version 2.0 (Hermes Medical Solutions, Stockholm, Sweden) software in these organs. The residence times for the skeletal system were assumed to be distributed equally between trabecular and cortical bone. An indirect bloodbased method using patient-based red marrow-to-blood ratio (RMBLR) and bone marrow mass was used to determine bone marrow self-dose [34,35].
Urinary excretion fraction at all time points was determined by applying the function A 0 (1 − e − ,T ). With a logarithmic function fit on the urinary excretion curve, effective excretion half-life was obtained. Using the total urinary excretion fraction, effective excretion half-life and 4-h voiding interval as input in Cloutier's dynamic urinary bladder model, residence time for urinary bladder contents was obtained. By subtracting residence times for kidneys, bone marrow, and skeletal system from whole body residence time, the remainder of body residence time was calculated.
Residence time for kidneys, cortical and trabecular bone, urinary bladder contents, red marrow self-dose, and remainder of body were used as an input in OLINDA/EXM version 2.0 (Hermes Medical Solutions, Stockholm, Sweden) software for calculation of organ absorbed doses and effective doses after adjusting the weight of patient organs by multiplying the reference adult male weight with factor obtained by dividing patient weight with the reference adult male weight. The mean of residence times and organ absorbed doses (mSv/MBq) were calculated. Figures 1 and 3 show the biodistribution of [ 177 Lu]Lu-DOTA ZOL in one patient with bronchial carcinoma and one patient with mCRPC, respectively. In the initial 20 min p.i. image data set, highest uptake was seen in the urinary bladder followed by kidneys and soft tissue with minimal uptake in the skeletal system. The kidneys showed a rapid decrease in activity at 3 h with minimum to no uptake after 24 h p.i. The intense uptake was seen in the skeletal system from 3 h onwards. Blood and soft tissue clearance and lesion to normal bone contrast increased in later images up to 168 h. The mean ± SD 24h whole body retention was found to be 31.25 ± 6.5.

Qualitative analysis
In this small patient study, we observed fast uptake and clearance kinetics of kidneys in patients with bronchial carcinoma compared with mCRPC patient, which resulted in better skeletal to soft tissue contrast as early as 3 h p.i. in the bronchial carcinoma patient compared with 24 h p.i. in mCRPC patient.

Quantitative analysis
Mean residence times (MBq-h/MBq) ( Table 2) was found to be highest in trabecular and cortical bone (31.9 h) followed by the remainder of the body (11.7 h), kidneys (1.84 h), urinary bladder (1.52 h), and bone marrow (0.03 h). In patient no. 1, residence times for the skeletal system and the kidney were lower compared with the other patients.

Discussion
Zoledronate presents as an ideal candidate for labeling with the therapeutic radionuclide lutetium-177 for radionuclide therapy of bone metastases, as it shows high osteoclast and hydroxyl apatite binding [27] and no in vivo biotransformation [3]. Preclinical small animal studies using [ 177 Lu]Lu-DOTA ZOL and [ 68 Ga]Ga-DOTA ZOL showed comparable results, suggesting the two tracers as new theranostic pair for bone-targeted radionuclide therapy [27].
Extrapolation of dosimetric analysis of [ 177 Lu]Lu-DOTA ZOL and [ 177 Lu]Lu-EDTMP from rats to humans revealed high kidney and trabecular bone absorbed doses as well as high trabecular bone to other organs absorbed dose ratios for [ 177 Lu]Lu-DOTA ZOL [28]. The higher thermodynamic and kinetic stability, leading to high bone uptake with low soft tissue accumulation, suggests [ 177 Lu]Lu-DOTA-ZOL to be a better therapeutic bisphosphonate compared with [ 177 Lu]Lu-EDTMP [2]. The current study is the first ever human biodistribution and dosimetric analysis for [ 177 Lu]Lu-DOTA ZOL in mCRPC and bronchial carcinoma patients. We noticed that the biodistribution of [ 177 Lu]Lu-DOTA ZOL in humans ( Figs. 1 and 3) is consistent with preclinical biodistribution studies in male Wistar rats [27,28]. We found the highest accumulation in the skeleton with fast kidney uptake and clearance. As the kidneys are the sole route of its excretion, the urinary bladder showed high uptake as well. Blood and soft tissue showed rapid clearance which resulted in good skeleton to soft tissue contrast. A rapid and biphasic blood clearance curve was found (Fig. 4) comparable to [ 177 Lu]Lu-EDTMP [5]. No uptake was seen in any other organ.
Prominent uptake in the skeletal system in bronchial carcinoma patients was visualized at 3 h p.i. image in contrast to 24 h p.i. in mCRPC patients. The finding of best bone-to-soft tissue contrast at 24 h p.i. in mCRPC patients is consistent with similar observations with [ 177 Lu]Lu-EDTMP distribution in mCRPC patients [5,7,26]. To establish whether the early uptake in bronchial carcinoma patients is a patient dependent or tumor dependent finding and can be of any significance in relation to tumor lesion doses needs further large scale and tumor lesion dosimetry studies.
The source organs identified for dosimetric analysis included the kidneys, bone marrow, urinary bladder, skeletal system, and the whole body. A biphasic kinetic behavior of [ 177 Lu]Lu-DOTA ZOL was observed in all source organs and the whole body. Hence, biexponential curve fitting was used for residence time calculations. Residence time was highest in the skeleton similar to [ 177 Lu]Lu-EDTMP (  [26]. The difference might be due to humerus [5] or femoral [26] activity extrapolation   [5,26]. This difference could be due to the use of Cloutier's method with cumulative urinary calculation from whole body retention and 4-h voiding intervals in our current study compared with the collection of urine samples for residence time calculation in the [ 177 Lu]Lu-EDTMP studies. [ 177 Lu]Lu-DOTA ZOL resulted in a lower bone marrow absorbed dose compared with [ 177 Lu]Lu-EDTMP [5,7,26], which in theory allows administration of higher therapeutic activities of [ 177 Lu]Lu-DOTA ZOL . Based on a maximum permissible radiation absorbed dose to the bone marrow of 2Gy, the maximum tolerated dose for [ 177 Lu]Lu-DOTA ZOL is estimated to be 3630-4980 MBq compared with 2000-3250 MBq for [ 177 Lu]Lu-EDTMP [5]. As a result, radiation absorbed dose of 11 to 16 Gy will be delivered to osteogenic cells by [ 177 Lu]Lu-DOTA ZOL which is comparable with 10.1 to 17.6 Gy for [ 177 Lu]Lu-EDTMP. Using these thresholds, the kidney absorbed dose remains well below the maximum permissible dose limit of 23 Gy.  As absorbed dose to kidneys is one of the important factors in radionuclide therapy using Lutetium-177labeled radiopharmaceuticals, we found that [ 177 Lu]Lu-DOTA ZOL delivers a lower (by a factor of 1.2 to 1.88) kidney dose (Fig. 5) in comparison with [ 177 Lu]Lu-PSMA-617 [33,36].
Further, we would like to add here that in this study we used complementary PET/CT images of the patient for organ and whole body thickness calculation. However, whole body SPECT/CT may result in similar images. Though time consuming and inconvenient for the seriously ill patients, these images can still be of added value in future dosimetry studies.

Conclusion
[ 177 Lu]Lu-DOTA ZOL is a promising new therapeutic radiopharmaceutical for radionuclide therapy of bone metastases due to excellent skeletal uptake, a lower low bone marrow dose than [ 177 Lu]Lu-EDTMP and a very low kidney dose. Labeling with gallium-68 delivers [ 68 Ga]Ga-DOTA ZOL , which represents an ideal theranostic counterpart for PET/CT imaging. Further studies are warranted to evaluate the efficacy and safety of radionuclide therapy with [ 177 Lu]Lu-DOTA ZOL in the clinical setting.