Open Access

Absorption, distribution and excretion of intravenously injected 68Ge/68Ga generator eluate in healthy rats, and estimation of human radiation dosimetry

  • Anu Autio1,
  • Helena Virtanen1,
  • Tuula Tolvanen1,
  • Heidi Liljenbäck1, 2,
  • Vesa Oikonen1,
  • Tiina Saanijoki1,
  • Riikka Siitonen1,
  • Meeri Käkelä1,
  • Andrea Schüssele3,
  • Mika Teräs1 and
  • Anne Roivainen1, 2, 4Email author
Contributed equally
EJNMMI Research20155:40

https://doi.org/10.1186/s13550-015-0117-z

Received: 15 April 2015

Accepted: 6 July 2015

Published: 17 July 2015

The Erratum to this article has been published in EJNMMI Research 2016 6:51

Abstract

Background

This study evaluated the absorption, distribution, and excretion of Gallium-68 (68Ga) radionuclide after a single intravenous (i.v.) injection of 68Ge/68Ga generator eluate in healthy rats. Additionally, human radiation doses were estimated from the rat data.

Methods

Twenty-one female and 21 male Sprague-Dawley rats were i.v. injected with 47 ± 4 MBq of 68Ge/68Ga generator eluate, and the radioactivity of excised organs was measured using a gamma counter at 5, 30, 60, 120, or 180 min afterwards (n = 3–7 for each time point). The radioactivity concentration and plasma pharmacokinetic parameters were calculated. Subsequently, the estimates for human radiation dosimetry were determined. Additionally, 4 female and 5 male rats were positron emission tomography (PET) imaged for in vivo visualization of biodistribution.

Results

68Ga radioactivity was cleared relatively slowly from blood circulation and excreted into the urine, with some retention in the liver and spleen. Notably, the 68Ga radioactivity in female genital organs, i.e., the uterus and ovaries, was considerable higher compared with male genitals. Extrapolating from the female and male rat 68Ga data, the estimated effective dose was 0.0308 mSv/MBq for a 57-kg woman and 0.0191 mSv/MBq for a 70-kg man.

Conclusions

The estimated human radiation burden of the 68Ge/68Ga generator eluate was slightly higher for females and similar for males as compared with somatostatin receptor ligands 68Ga-DOTANOC, 68Ga-DOTATOC, and 68Ga-DOTATATE, which is probably due to the retention in the liver and spleen. Our results revealed some differences between female and male rat data, which, at least in part, may be explained by the small sample size.

Keywords

68Ge/68Ga generatorDosimetryRatWhole-body distribution

Background

Gallium-68 (68Ga)-labeled tracers are increasingly used in positron emission tomography (PET) for diagnostic purposes. The prototypes of 68Ga-labeled PET imaging agents are the somatostatin receptor ligands, 68Ga-DOTATOC/NOC/TATE peptides, which are today routinely used for PET imaging of neuroendocrine tumors [13]. Currently, many other 68Ga-labeled peptide families are under clinical evaluation, such as bombesins (gastrin-releasing peptide receptor ligands), exendins (glucagon-like peptide 1 receptor ligands), and arginine-glycine-aspartic acids (RGDs) (integrin receptor ligands). In addition to oncology, 68Ga-tracer-based PET has been studied for imaging of inflammation [4].

68Ga offers a cyclotron-independent, convenient, and low-cost access to PET imaging agents. It is readily available by elution from a 68Germanium/68Gallium (68Ge/68Ga) generator possessing a 1-year life span depending on the uploaded 68Ge radioactivity. Furthermore, 68Ga has several convenient characteristics, such as β+ decay 89 %, Eβ+ max 1.9 MeV, and a sufficiently long half-life (67.71 min) for PET imaging.

The purpose of this study was to obtain preclinical information to support the use of the 68Ge/68Ga generator eluate for medical application. The absorption, distribution, and excretion of radioactivity after a single intravenous injection of the 68Ge/68Ga generator eluate were assessed in order to determine the radiation dosimetry of 68Ga. This study investigated the possible consequences of poor radiolabeling efficiency or in vivo dissociation of the radiolabeled conjugate, i.e., issues related to the effects produced in the patient by the free radionuclide. When 68Ga is eluted from 68Ge/68Ga generator with 0.1 mol/l hydrochloric acid solution, it is in the form of 68GaCl3. In aqueous solution; 68Ga is in the form of the hydrated gallium ion [Ga(H2O)6]3+. Insoluble neutral hydroxide colloids 68Ga(OH)3, may precipitated depending on pH (>4) and the concentration of 68Ga. After intravenous injection, the 68Ga radioactivity can migrate in the blood circulation as free 68Ga3+ or 68Ga3+ bound to transferrin, ferritin, or lactoferrin. Here, the absorption, distribution, and excretion of 68Ga radioactivity after a single intravenous (i.v.) injection were studied in healthy, mature Sprague-Dawley rats up to 3 h, and the estimates for human radiation dosimetry were calculated.

Methods

All animal experiments were approved by the National Animal Experiment Board in Finland (ELLA) and the Regional State Administrative Agency for Southern Finland (ESAVI) and conducted in accordance with the relevant European Union Directive. This preclinical study was performed without randomization and blinding. The healthy, mature Sprague-Dawley rats were purchased from Harlan, The Netherlands, and they were of specific pathogen free (SPF) quality. The rats were left to acclimate for a minimum of 5 days after their arrival before the study. They were housed at room temperature (18–24 °C) and relative humidity of 40–70 %. Artificial lighting was used, with 12 h of light (6 a.m. to 6 p.m.) and 12 h of dark (6 p.m. to 6 a.m.). The animals received regular feed, and tap water was offered ad libitum. They fasted for 4–6 h prior to the administration of the 68Ge/68Ga generator eluate.

68Ge/68Ga generator eluate

The 68Ge/68Ga generator (Eckert-Ziegler Source No. 1484-7 with 1850 MBq nominal radioactivity at reference date) was eluted with 6 ml of 0.1 mol/l hydrochloric acid; the 0.7−1.2 ml radioactive elution peak was collected and diluted with phosphate-buffered saline (PBS) (600−860 μl, pH 7 ± 0) for i.v. injection.

Biodistribution

Twenty-one female (weight 248 ± 13 g) and 21 male rats (weight 342 ± 47 g) were examined at five different time points post-injection (5, 30, 60, 120, and 180 min), with 3–7 female and 3–7 male rats per time point. The animals were placed in an immobilizer (AgnTho’s AB, Lidingö, Sweden), and a catheter was inserted in their tail vein. The rats were i.v. injected with 47 ± 4 MBq of 68Ge/68Ga generator eluate as a bolus and promptly flushed with physiological saline after injection. Animals were killed with an overdose of pentobarbital (Mebunat, Orion Pharma, Finland). Various organs were excised, weighed, and measured for total radioactivity by using a gamma counter (1480 Wizard 3" PerkinElmer/Wallac, Turku, Finland) cross-calibrated with the dose calibrator (VDC-404; Veenstra Instruments, Joure, The Netherlands). Of blood, bone, bone marrow, brown adipose tissue, fat, plasma, skeletal muscle, skin, and urine, only small samples were taken and measured for radioactivity and weight. All other tissues and organs were measured in their entirety. The residual carcass was assessed with a dose calibrator. Urine was obtained directly from the urinary bladder using a needle and syringe, and total radioactivity was measured as described above. Blood was obtained by means of cardiac puncture. Radioactivity of whole blood was measured. Plasma was separated by centrifugation (2118×g for 5 min at 4 °C), and plasma radioactivity was measured. The radioactivity concentration was decay corrected to the time of injection, and the results were expressed as standardized uptake values (SUV) and percentage of injected radioactivity dose per gram of tissue (%ID/g).

In addition to ex vivo studies, nine animals (five males 309–355 g, four females 245–287 g) were PET imaged for 180 min to visualize whole-body distribution in vivo and to obtain time-activity curves. The animals were anesthetized with isoflurane (induction 3 % and maintenance 1.7 %), and a catheter was inserted in their tail vein. The rats were i.v. injected with 45 ± 3 MBq of 68Ge/68Ga generator eluate and imaged by using a High Resolution Research Tomograph (HRRT; Siemens Medical Systems, Knoxville, TN, USA) PET camera. Both rats were imaged at the same time. They were kept on a warm pallet during the imaging procedure. For attenuation correction, a 6-min transmission scan was obtained using a collimated transmission point source. The PET imaging data were reconstructed using the ordered-subsets expectation maximization 3D algorithm (OSEM3D) with attenuation correction based on transmission source measurement.

Plasma pharmacokinetics

Plasma pharmacokinetic parameters, i.e., the area under the curve (AUC), elimination rate constant (kel), total clearance (ClT), and half-life (t1/2), were calculated using Microsoft Excel from the plasma concentrations at 5, 30, 60, 120, and 180 min after tracer injection. Only one sample could be obtained from each animal, wherefore the sampled concentrations from all animals (males and females separately) were combined in order to produce the curve of radioactivity concentration vs. time after injection. Since the injected radioactive dose per weight was not exactly the same in all animals, the plasma SUVs (radioactivity concentrations corrected by injected dose and animal weight) were used for calculation. Initial concentration (C 0) was estimated by back-extrapolating from the log-linear regression of the two first concentration values (5- and 30-min samples). AUC between 0 and 180 min was calculated using the linear trapezoidal rule, starting from C 0. Log-linear regression of the last three concentrations (60-, 120-, and 180-min samples) was used to estimate the AUC from 180 min to infinity that is, to calculate AUC0–∞ and to estimate kel and t1/2. Because plasma concentrations were given in SUV units, the total clearance (CIT) is calculated as 1/AUC0–∞, and the unit of CIT is then (g plasma/(g rat × min)).

Estimation of human radiation dose

Absorbed doses were calculated with the OLINDA/EXM version 1.0 software (organ level internal dose assessment and exponential modeling; Vanderbilt University, Nashville, TN, USA), which applies the MIRD schema (developed by the Medical Internal Radiation Dose committee of the Society of Nuclear Medicine) for radiation dose calculations in internal exposure. The software includes radionuclide information and selection of human body phantoms. The residence times derived from the rat data were integrated as the area under the time-activity curve. The residence times were converted into corresponding human values by multiplication with a factor to scale the organ and body weights: (WTB,rat/WOrgan,rat) × (WOrgan,human/WTB,human), where WTB,rat and WTB,human are the body weights of rat and human (a 57-kg female or 70-kg male), respectively; and WOrgan,rat and WOrgan,human are the organ weights of rat and human (organ weights for a 57-kg female or 70-kg male), respectively.

Statistical analysis

The mean values are calculated from the individual measurements and expressed with an accuracy of one standard deviation (mean ± SD). Differences between genders were assessed with Student’s t test.

Results

Animal and tissue/organ weights and ex vivo biodistribution data from the rats are summarized in Tables 1 and 2, and 180-min results visualized in Fig. 1. 68Ga radioactivity was slowly cleared from blood circulation and excreted predominantly into the urine, with some retention in the liver and kidneys. Interestingly, the 68Ga radioactivity in female genital organs, i.e., the uterus and ovaries, was considerable higher than in male genitals (ovaries 0.750 %ID/g, uterus 0.941, testes 0.198 at 180 min). Plasma had also large difference 2.865 %ID/g (female) vs. 1.544 (male) at 180 min. In vivo PET images were in the line with ex vivo measurements (Fig. 2). Estimated plasma pharmacokinetic parameters for 68Ga radioactivity are given in Table 3. Plasma concentration was relatively high at the final time point (180 min), which may lead in uncertainty in the estimation of AUC0–∞.
Table 1

Organ weights and ex vivo biodistribution of 68Ga/68Ge generator eluate in female rats

Tissue/organ

Weight (g)

5 min

30 min

60 min

120 min

180 min

Adrenal glands

0.067 ± 0.016

0.673 ± 0.111

0.603 ± 0.271

0.705 ± 0.264

0.567 ± 0.226

0.497 ± 0.184

Blood

a

2.909 ± 0.272

2.509 ± 1.359

2.563 ± 0.909

2.128 ± 0.953

1.668 ± 0.428

Bone (femur, both)

a

0.298 ± 0.071

0.589 ± 0.132

0.820 ± 0.396

1.001 ± 0.363

0.963 ± 0.370

Bone marrow (femur, both)

a

0.586 ± 0.133

0.296 ± 0.051

0.705 ± 0.242

0.790 ± 0.366

0.710 ± 0.231

Brain

1.612 ± 0.103

0.075 ± 0.006

0.079 ± 0.040

0.084 ± 0.025

0.080 ± 0.041

0.052 ± 0.016

Brown adipose tissue

a

0.405 ± 0.017

0.605 ± 0.229

0.471 ± 0.213

0.376 ± 0.201

0.316 ± 0.126

Colon (without contents)

1.069 ± 0.288

0.289 ± 0.095

0.668 ± 0.283

0.615 ± 0.337

0.531 ± 0.302

0.431 ± 0.277

Fat (intraperitoneal)

a

0.058 ± 0.028

0.177 ± 0.093

0.145 ± 0.073

0.158 ± 0.151

0.099 ± 0.038

Heart

0.801 ± 0.049

0.723 ± 0.115

0.755 ± 0.462

0.617 ± 0.235

0.540 ± 0.204

0.439 ± 0.141

Ileum (without contents)

3.322 ± 1.420

0.271 ± 0.040

0.550 ± 0.191

0.465 ± 0.070

0.631 ± 0.426

0.520 ± 0.202

Kidneys

1.389 ± 0.162

0.773 ± 0.173

0.875 ± 0.328

0.823 ± 0.301

0.722 ± 0.324

0.679 ± 0.208

Liver

7.179 ± 0.836

1.225 ± 0.374

1.529 ± 0.450

1.490 ± 0.516

1.597 ± 0.627

1.178 ± 0.759

Lungs

1.117 ± 0.079

0.949 ± 0.212

1.078 ± 0.520

1.136 ± 0.379

0.894 ± 0.450

0.754 ± 0.268

Ovaries

0.135 ± 0.024

0.574 ± 0.174

0.784 ± 0.429

1.343 ± 1.257

0.782 ± 0.286

0.750 ± 0.124

Pancreas

1.180 ± 0.225

0.464 ± 0.283

0.389 ± 0.211

0.364 ± 0.174

0.331 ± 0.164

0.346 ± 0.233

Plasma

a

5.083 ± 0.486

4.491 ± 2.419

4.451 ± 1.523

3.742 ± 1.602

2.865 ± 0.813

Salivary glands

0.496 ± 0.047

0.397 ± 0.099

0.630 ± 0.230

0.506 ± 0.178

0.485 ± 0.229

0.382 ± 0.165

Skeletal muscle

a

0.132 ± 0.048

0.253 ± 0.080

0.214 ± 0.118

0.190 ± 0.078

0.143 ± 0.077

Skin

a

0.096 ± 0.029

0.341 ± 0.142

0.312 ± 0.176

0.265 ± 0.089

0.263 ± 0.079

Spleen

0.687 ± 0.136

0.493 ± 0.137

0.836 ± 0.204

0.785 ± 0.269

0.687 ± 0.201

0.661 ± 0.258

Stomach (without contents)

1.184 ± 0.216

0.261 ± 0.060

0.384 ± 0.117

0.471 ± 0.196

0.441 ± 0.255

0.369 ± 0.159

Thymus

0.305 ± 0.082

0.234 ± 0.003

0.259 ± 0.143

0.225 ± 0.089

0.264 ± 0.193

0.181 ± 0.106

Thyroids

0.017 ± 0.005

0.516 ± 0.020

0.656 ± 0.327

0.607 ± 0.245

0.576 ± 0.347

0.473 ± 0.174

Urinary bladder (without contents)

0.068 ± 0.021

0.256 ± 0.080

0.442 ± 0.172

0.636 ± 0.311

0.734 ± 0.187

0.469 ± 0.187

Urine

a

0.417 ± 0.134

13.162 ± 3.607

3.526 ± 1.783

5.228 ± 2.368

2.314 ± 0.981

Uterus

0.591 ± 0.153

0.361 ± 0.145

1.023 ± 0.482

1.149 ± 0.865

0.819 ± 0.553

0.941 ± 0.585

Residual carcass

208.418 ± 11.160

0.200 ± 0.010

0.318 ± 0.036

0.266 ± 0.025

0.269 ± 0.023

0.262 ± 0.029

Results are expressed as percentage of injected radioactivity dose per gram of tissue (mean ± SD)

aOf blood, bone, bone marrow, brown adipose tissue, fat, plasma, skeletal muscle, skin, and urine, only a small sample was taken and weighed. All other tissues/organs were measured in their entirety

Table 2

Tissue/organ weights and ex vivo biodistribution of 68Ga/68Ge generator eluate in male rats

Tissue/organ

Weight (g)

5 min

30 min

60 min

120 min

180 min

Adrenal glands

0.040 ± 0.012

0.635 ± 0.404

0.303 ± 0.047

0.338 ± 0.040

0.355 ± 0.174

0.278 ± 0.011

Blood

a

1.556 ± 0.476

1.119 ± 0.088

1.175 ± 0.128

0.970 ± 0.165

0.908 ± 0.157

Bone (femur, both)

a

0.285 ± 0.100

0.387 ± 0.188

0.500 ± 0.176

0.657 ± 0.240

0.854 ± 0.256

Bone marrow (femur, both)

a

0.494 ± 0.061

0.354 ± 0.020

0.423 ± 0.066

0.394 ± 0.123

0.490 ± 0.064

Brain

1.633 ± 0.163

0.078 ± 0.020

0.041 ± 0.003

0.048 ± 0.006

0.034 ± 0.002

0.035 ± 0.007

Brown adipose tissue

a

0.294 ± 0.099

0.279 ± 0.122

0.318 ± 0.075

0.255 ± 0.071

0.217 ± 0.070

Colon (without contents)

1.223 ± 0.424

0.230 ± 0.018

0.260 ± 0.048

0.285 ± 0.038

0.227 ± 0.077

0.305 ± 0.066

Fat (intraperitoneal)

a

0.050 ± 0.019

0.069 ± 0.013

0.090 ± 0.031

0.069 ± 0.046

0.053 ± 0.009

Heart

1.086 ± 0.113

0.494 ± 0.075

0.338 ± 0.070

0.320 ± 0.032

0.270 ± 0.053

0.251 ± 0.042

Ileum (without contents)

3.923 ± 1.768

0.205 ± 0.088

0.186 ± 0.022

0.268 ± 0.074

0.222 ± 0.114

0.308 ± 0.081

Kidneys

2.113 ± 0.279

0.993 ± 0.730

0.359 ± 0.070

0.404 ± 0.026

0.343 ± 0.119

0.397 ± 0.025

Liver

12.497 ± 1.444

1.044 ± 0.516

0.514 ± 0.195

0.770 ± 0.336

0.746 ± 0.421

0.498 ± 0.209

Lungs

1.277 ± 0.133

0.608 ± 0.028

0.479 ± 0.045

0.484 ± 0.196

0.455 ± 0.079

0.374 ± 0.037

Pancreas

1.307 ± 0.301

0.246 ± 0.042

0.207 ± 0.067

0.212 ± 0.031

0.181 ± 0.034

0.173 ± 0.016

Plasma

a

3.481 ± 0.980

2.078 ± 0.194

2.162 ± 0.251

1.639 ± 0.259

1.544 ± 0.203

Salivary glands

0.596 ± 0.060

0.351 ± 0.079

0.304 ± 0.018

0.336 ± 0.058

0.331 ± 0.075

0.315 ± 0.034

Skeletal muscle

a

0.085 ± 0.069

0.125 ± 0.036

0.142 ± 0.019

0.151 ± 0.028

0.123 ± 0.023

Skin

a

0.124 ± 0.074

0.157 ± 0.060

0.248 ± 0.038

0.204 ± 0.054

0.219 ± 0.037

Spleen

0.864 ± 0.135

0.520 ± 0.114

0.383 ± 0.099

0.516 ± 0.193

0.582 ± 0.255

0.455 ± 0.129

Stomach (without contents)

1.403 ± 0.241

0.222 ± 0.045

0.227 ± 0.061

0.268 ± 0.036

0.250 ± 0.058

0.264 ± 0.048

Testes

3.509 ± 0.214

0.076 ± 0.054

0.075 ± 0.023

0.140 ± 0.020

0.175 ± 0.026

0.198 ± 0.018

Thymus

0.497 ± 0.088

0.190 ± 0.033

0.137 ± 0.055

0.182 ± 0.050

0.141 ± 0.051

0.154 ± 0.064

Thyroids

0.020 ± 0.007

0.536 ± 0.040

0.355 ± 0.057

0.403 ± 0.131

0.415 ± 0.106

0.370 ± 0.105

Urinary bladder (without contents)

0.070 ± 0.015

0.267 ± 0.177

0.553 ± 0.360

0.502 ± 0.191

0.478 ± 0.216

0.418 ± 0.259

Urine

a

0.758 ± 0.727

1.342 ± 0.530

1.408 ± 0.736

3.111 ± 3.927

1.520 ± 0.894

Residual carcass

289.423 ± 45.504

0.177 ± 0.082

0.218 ± 0.051

0.207 ± 0.039

0.215 ± 0.048

0.206 ± 0.038

Results are expressed as percentage of injected radioactivity dose per gram of tissue (mean ± SD)

aOf blood, bone, bone marrow, brown adipose tissue, fat, plasma, skeletal muscle, skin, and urine, only a small sample was taken and weighed. All other tissues/organs were measured in their entirety

Fig. 1

Ex vivo biodistribution of 68Ga radioactivity in rats at 180 min post-injection. Statistically significant (P < 0.05) differences between female and male are marked as asterisk

Fig. 2

PET images and time-activity curves. Left panels: Healthy Sprague-Dawley rats were intravenously injected with 68Ge/68Ga generator eluate. Images are summations from 0–180 min after injection and displayed in the same color scale as percentage of injected dose per gram of tissue (%ID/g). Male rat: 350 g, 50 MBq (0.007 MBq/kg); female rat 259 g, 48 MBq (0.005 MBq/kg). Right panels: Mean time-activity curves of 68Ga radioactivity in selected organs/tissues of male (upper) and female (lower) rats. Error bars denote standard deviation. According to Student’s t test, the differences in blood (P = 0.00026) and testes vs. uterus (P < 0.0001) were statistically significant at 180-min post-injection. The “blood” values were obtained from heart left ventricle

Table 3

Plasma pharmacokinetic parameters for 68Ga radioactivity after an intravenous bolus injection of 68Ga/68Ge generator eluate in rats

Parameter

Males (n = 21)

Females (n = 21)

kel (1/h)

0.175349

0.201682

t1/2 (h)

3.952956

3.436839

AUC0–∞ (h × g/g)

48.66309

65.64807

ClT (g plasma/(g rat × h))

0.020549

0.015233

AUC area under the curve, k el elimination rate constant, Cl T total clearance, C 0 initial concentration, t 1/2 plasma half-life

The human residence times for the various source organs and radiation dose estimates for 68Ga radioactivity, extrapolated from rat biodistribution data, are listed in Table 4. The estimations of the absorbed doses were calculated for a 70-kg adult male and a 57-kg adult female. Extrapolating from the female rat data, the effective dose for a 57-kg adult female was 0.0308 mSv/MBq, i.e., 7.7 mSv from an intravenously injected radioactivity of 250 MBq. The corresponding estimates for 15-, 10-, 5-, and 1-year-old and newborn females are presented in Table 5. Estimated from female rat data, the absorbed doses were the greatest in heart wall (0.501 mSv/MBq), osteogenic cells (0.076 mSv/MBq), liver (0.072 mSv/MBq), lungs (0.0502 mSv/MBq), and spleen (0.034 mSv/MBq). Extrapolating from the male rat data, the effective dose for a 70-kg adult male was 0.0191 mSv/MBq, i.e., 4.8 mSv from an intravenously injected radioactivity of 250 MBq. The corresponding estimates for 15-, 10-, 5-, and 1-year-old and newborn males are presented in Table 6. Estimated from the male rat data, the absorbed doses were greatest in heart wall (0.216 mSv/MBq), liver (0.0652 mSv/MBq), osteogenic cells (0.0418 mSv/MBq), urinary bladder wall (0.0382 mSv/MBq), and lungs (0.0245 mSv/MBq).
Table 4

Human residence times and radiation dose estimates for 68Ga radioactivity extrapolated from the rat biodistribution data

Organ

Residence time (h)

Dose (mSv/MBq)

 

Female rat data

Male rat data

Female rat data

Male rat data

Adrenals

  

0.02210

0.01600

Blood

0.7790

0.3708

  

Bone

0.3120

0.1950

  

Bone marrow

0.0688

0.0425

  

Brain

0.0064

0.0043

0.00591

0.00372

Breasts

  

0.01790

0.01260

Gallbladder wall

  

0.01950

0.01680

Lower large intestine wall

  

0.01520

0.01260

Small intestine

  

0.01480

0.01350

Stomach wall

  

0.01870

0.01400

Upper large intestine wall

  

0.01580

0.01360

Heart wall

0.0117

0.0072

0.50100

0.21600

Kidneys

0.0149

0.0118

0.03360

0.02430

Liver

0.1716

0.2226

0.07220

0.06520

Lungs

0.0591

0.0352

0.05020

0.02450

Muscle

0.4238

0.2484

0.01900

0.00928

Ovaries

0.0006

 

0.03080

0.01290

Pancreas

0.0027

0.0013

0.02860

0.01530

Red marrow

0.0688

0.0425

0.02530

0.01850

Osteogenic cells

  

0.07580

0.04180

Skin

  

0.01170

0.0097300

Spleen

0.0081

0.0069

0.03370

0.02280

Thymus

  

0.03050

0.01860

Thyroid

0.0008

0.0005

0.02640

0.02100

Testes

 

0.0005

 

0.00934

Urinary bladder wall

  

0.01350

0.03820

Uterus

0.0006

 

0.01140

0.01320

Whole body

  

0.02560

0.01730

Effective dose

  

0.0308

0.0191

Table 5

Additional dosimetry estimates based on a female rat distribution data

 

Absorbed dose per unit radioactivity administered (mSv/MBq)

Organ

15-year-olds

10-year-olds

5-year-olds

1-year-olds

Newborn

(50 kg)

(30 kg)

(17 kg)

(10 kg)

(5 kg)

Adrenals

0.0220

0.0337

0.0521

0.0922

0.212

Brain

0.00526

0.00685

0.00531

0.0148

0.0333

Breasts

0.0178

0.0304

0.0483

0.0885

0.211

Gallbladder wall

0.0194

0.0307

0.0466

0.0875

0.217

Lower large intestine wall

0.0144

0.0236

0.0374

0.0696

0.176

Small intestine

0.0157

0.0251

0.0400

0.0762

0.182

Stomach wall

0.0187

0.0285

0.0451

0.0849

0.202

Upper large intestine wall

0.0153

0.0247

0.0400

0.0754

0.185

Heart wall

0.576

0.893

1.44

2.61

5.11

Kidneys

0.0363

0.0521

0.0777

0.139

0.351

Liver

0.0722

0.109

0.164

0.315

0.721

Lungs

0.0575

0.0823

0.125

0.239

0.611

Muscle

0.0201

0.0382

0.109

0.215

0.305

Ovaries

0.0349

0.0893

0.1550

0.349

0.710

Pancreas

0.0335

0.0608

0.0823

0.166

0.496

Red marrow

0.0262

0.0455

0.0828

0.201

0.737

Osteogenic cells

0.0745

0.117

0.194

0.452

1.39

Skin

0.0116

0.0187

0.0306

0.0595

0.151

Spleen

0.0391

0.0602

0.0955

0.174

0.449

Testes

0.0126

0.0203

0.0327

0.0637

0.156

Thymus

0.0311

0.0413

0.0622

0.107

0.237

Thyroid

0.0342

0.0537

0.113

0.213

0.314

Urinary bladder wall

0.0733

0.113

0.180

0.345

0.895

Uterus

0.0118

0.0721

0.110

0.198

0.136

Total body

0.0258

0.0420

0.0678

0.133

0.338

Effective dose (mSv/MBq)

0.03110

0.0582

0.096800

0.20100

0.48500

Table 6

Additional dosimetry estimates based on a male rat distribution data

 

Absorbed dose per unit radioactivity administered (mSv/MBq)

Organ

15-year-olds

10-year-olds

5-year-olds

1-year-olds

Newborn

(50 kg)

(30 kg)

(17 kg)

(10 kg)

(5 kg)

Adrenals

0.0201

0.0313

0.0491

0.0897

0.231

Brain

0.0039

0.00523

0.00723

0.0115

0.0259

Breasts

0.01580

0.0258

0.0416

0.0790

0.195

Gallbladder wall

0.0203

0.0312

0.0483

0.0921

0.228

Lower large intestine wall

0.00149

0.0248

0.0396

0.0746

0.191

Small intestine

0.0167

0.0267

0.0427

0.0818

0.199

Stomach wall

0.0175

0.0275

0.0440

0.0843

0.206

Upper large intestine wall

0.0165

0.0266

0.0428

0.0814

0.202

Heart wall

0.280

0.435

0.700

1.27

2.49

Kidneys

0.0295

0.0421

0.0633

0.113

0.282

Liver

0.0876

0.133

0.199

0.384

0.888

Lungs

0.0352

0.0504

0.0765

0.147

0.374

Muscle

0.0137

0.0253

0.0665

0.134

0.199

Ovaries

0.0175

0.0258

0.0461

0.0799

0.194

Pancreas

0.02060

0.0361

0.0501

0.098

0.275

Red marrow

0.02190

0.0355

0.0643

0.1620

0.630

Osteogenic cells

0.05470

0.0850

0.1390

0.3220

0.974

Skin

0.0121

0.0198

0.0326

0.0639

0.164

Spleen

0.0327

0.0503

0.0801

0.1460

0.378

Testes

0.0185

0.1120

0.1320

0.1810

0.269

Thymus

0.02270

0.0324

0.0501

0.0911

0.214

Thyroid

0.0330

0.0514

0.11

0.206

0.298

Urinary bladder wall

0.0488

0.0760

0.122

0.232

0.6040

Uterus

0.0165

0.0262

0.0424

0.0810

0.197

Total body

0.0220

0.0360

0.0584

0.1150

0.292

Effective dose (mSv/MBq)

0.02530

0.05580

0.081000

0.14600

0.34300

Discussion

The latest research has produced a series of new compounds labeled with 68Ga, which is a positron-emitting metal especially suitable for labeling of peptides and PET. In this study, we investigated the absorption, distribution, and excretion of 68Ga/68Ge radioactivity after a single intravenous injection of 68Ge/68Ga generator eluate in female and male rats over a period of 3 h.

The observed differences between the female and male biodistribution data are probably, at least in part, due to the small number of animals. However, higher concentrations of some elements, including iron, have been observed in the organs of female rats than in male rats, and since Ga3+ is transported into tissues in a similar way as Fe3+, this may explain part of the gender difference observed in this study [5]. This, in addition to higher injected radioactivity dose per gram, might in part explain higher plasma values of female rats. Interestingly, the estimated human radiation dose was higher when using the female rat data than when using the male rat data. Possible explanations for this include the small sample size and/or actual gender dependent differences in handling certain elements [5]. The body weight of female rats was approximately 100 g lower than that of male rats. However, both genders received an identical 50-MBq dose of 68Ga eluate. This may also be one source of difference.

When the studies started, the 68Ge/68Ga generator was already used for 7 month; the studies lasted up to 5 month. According to manufacturer’s metals screen by inductively coupled plasma mass spectrometry (ICP-MS), the 68Ge/68Ga generator (no. 1484-7, with 1850-MBq nominal radioactivity) had antimony 0.004 ppm, boron 0.17 ppm, sodium 0.56 ppm, titanium 0.069 ppm, and zinc 0.006 ppm, which reflects the typical metal impurities in the eluate. In the respective Ph. Eur. monograph, only zinc and iron are mentioned as metal impurities, because they might interfere as 3+ metals with the Ga-68 during the labeling. The limit of zinc and iron is 10 μg/GBq. The generator has a zinc level of 0.02 μg/GBq, which is well below the limit, and iron was not detected by ICP-MS by a detection limit of 0.006 ppm, which also correlates to a value way below the limit. The 68Ge breakthrough was 0.000017 % correlated to the 68Ga radioactivity of the eluate at the reference date. This value is well below the limit of 68Ge-breakthrough mentioned in the respective Ph. Eur. monograph. Thus, it was estimated that these levels of metal impurities and 68Ge breakthrough had no effect on the results. However, the chemical form of 68Ga used in this study (68Ga eluate was mixed with a phosphate-buffered saline) might be different from 68Ga-species present as by-products in 68Ga-labeled radiopharmaceuticals. In radiolabeling peptides, 68Ga is reacted with chelate-conjugated peptides at elevated temperature, which should accelerate the hydrolysis reaction of Ga that does not form complex with peptide-chelate conjugate.

The whole-body distribution of 68Ga radioactivity reported here is in line with previous publications. Velikyan and co-workers studied biodistribution of 68GaCl3 in healthy Sprague-Dawley rats in order to control the organs where the accumulation would occur in case of impure tracer or in vivo release of 68Ga from the tracer (68Ga-DOTATOC and 68Ga-DOTATATE). The 68GaCl3 was acetate buffered to pH of 4.6 and formulated with phosphate-buffered saline (pH 7.4) for i.v. injection. The 68Ga radioactivity concentration at 75-min post-injection was the highest in the blood, and the accumulation in the heart, lung, liver, and spleen was considerably higher as compared to that of peptide tracers [6]. Previously, we i.v. injected NaOH neutralized 68Ga-chloride (12 MBq from Cyclotron Co. 68Ge/68Ga generator, Obninsk, Russia) in anesthetized, athymic, male Hsd/RH-rnu/rnu rats having subcutaneous tumor xenografts and reported the following SUVs at 90 min after injection (the values at 120 min of the present report are given in the parentheses): blood 2.7 ± 0.3 (3.074 ± 0.337), liver 5.9 ± 3.3 (2.280 ± 0.918), lung 1.9 ± 0.8 (1.445 ± 0.195), muscle 0.2 ± 0.03 (0.478 ± 0.067), and skin 0.5 ± 0.1 (0.662 ± 0.233) [7]. Subsequently, we also studied distribution of 68GaCl3 (Cyclotron Co., Obninsk, Russia) in healthy C57BL/6 N mice. The 68GaCl3 was neutralized with 1 mol/l sodium hydroxide to pH of 7, and the final product contained 13 % of colloidal forms of 68Ga as determined by ultrafiltration. Still, the biodistribution of 68Ga radioactivity was quite similar to the present study. The highest level of 68Ga radioactivity at 3-h post-injection was found in the blood and liver followed by spleen, kidneys, bone with bone marrow, and lung, respectively [8]. Nanni and co-workers have studied 68Ga-citrate in patients with infectious diseases [9]. Since citrate is only a weak chelator of 68Ga, the radionuclide is rapidly released in vivo and subsequently binds to transferrin and some other plasma proteins. The biodistribution of 68Ga-citrate may actually resemble that of free 68Ga or the eluate of the 68Ge/68Ga generator. In clinical whole-body PET scanning, 68Ga-citrate showed relatively high vascular radioactivity, moderate hepatic uptake, mild bone marrow radioactivity, and no bowel radioactivity. The relatively high vascular radioactivity, which is not seen in 67Ga-citrate scintigraphy, was a particularly interesting finding. In the current rat study, the elimination of radioactivity in urine and feces at each time point and the overall mass balance as a percentage of administered radioactivities could not be determined since urine or feces were sampled, not collected in their entirety. The observed high plasma values in rats supports that the 68Ga radioactivity is bound to transferrin.

68Ga has been used extensively for the labeling of synthetic peptides. However, there are only few human dosimetry reports available, including, for example, those on peptide analogues that bind to somatostatin receptors (Table 7) [1015]. The effective dose of the 68Ge/68Ga generator eluate reported here is somewhat higher than that of 68Ga-DOTANOC and 68Ga-DOTATOC [10, 11]. The higher dose of the 68Ge/68Ga generator eluate can be explained by the slow clearance from the blood and the retention in the liver. The biodistribution of 68Ga-labeled complexes is determined by the pharmacokinetics of the complexing molecules, such as peptides, and not by the incorporated Ga3+.
Table 7

Human effective doses of 68Ga radiopharmaceuticals and 18 F-FDG

Radiopharmaceutical

Effective dose (mSv/MBq)

Reference

68Ga-DOTANOC

0.025

Pettinato 2008 [10]

68Ga-DOTATOC

0.023

Hartmann 2009 [11]

68Ga-DOTATATE

0.021

Sandström 2013 [12]

68Ga-NOTA-2Rs15d

0.0218a

Xavier 2013 [13]

BAY86-7548

0.051

Roivainen 2013 [14]

18F-FDG

0.0190

ICRP Publication 1998 [15]

68Ga-eluate

0.0308b, 0.0191c

Present study

aExtrapolated from tumor xenograft mice

bObtained by using female rat data

cObtained by using male rat data

The radiation dose resulting from the i.v. injection of 68Ga-citrate has been estimated from 67Ga-citrate data [16]. The estimated absorbed dose for total body, calculated assuming a uniform distribution of radioactivity, was 0.052 rads/mCi. For 68Ge/68Ga generator eluate, the total-body radiation dose estimates for female and male rats were 0.02560 and 0.01730 mSv/MBq, respectively. However, the 68Ga-citrate and 68Ga-eluate values are not directly comparable because they are expressed with different units (rads/mCi vs. mSv/MBq), but assuming that only gamma radiation is taken into account for the energy dose, the rad value can be converted into the equivalent dose value rem, thus giving an absorbed dose of 0.052 rem/mCi = 0.014 mSv/MBq. There are limited number of studies comparing the PET tracer dosimetry in animals and humans. Table 8 contains a list of reference studies in which human effective doses derived from preclinical studies are reported and compared to effective doses from human measurements [1724]. The absorbed doses and effective doses from the in vivo studies in rats may be different from those obtained in human studies because of the dissimilar physiology of rodents and humans. For example, the blood flow rate has a remarkable influence on the time-activity curve shape, the area under the curve, and the measured number of disintegrations.
Table 8

Comparison of preclinical studies with the human effective dose (ED) estimates

PET tracer

Animal derived ED (mSv/MBq)

Human derived ED (mSv/MBq)

Reference

11C-6-OH-BTA-1

0.0065a

0.0045

Parsey 2005; Scheinin 2007 [17, 18]

11C-MPGA

0.0048

0.0053

Santens 1998 [19]

6-18 F-Fluoro-L-Dopa

0.0539b

0.0199

Harvey 1985; Brown 1998 [20, 21]

18 F-FET

0.0186

0.0165

Tang 2003; Pauleit 2003 [22, 23]

11C-Choline

0.0028

0.0044

Tolvanen 2010 [24]

aMales only

bED value calculated with the biological risk weight factors in accordance with ICRP 30 publication

In this study, scaling between rat and human data was performed using the overall non-organ-specific weight. In general, interspecies extrapolation of biokinetic data is based on the fact that the cellular structures and biochemistry are remarkably alike across the entire animal kingdom. Despite these similarities, however, the extrapolation of biokinetic data from laboratory animals to humans entails uncertainty, particularly for the liver, due to the qualitative differences among various species in the handling of many elements by this organ. Allometric scaling from laboratory animals to humans on the basis of body weight or surface area is the most commonly used method. It is based on the assumption that the biokinetics of compounds primarily depends on the metabolic rate of the animal and that the metabolic rate is a function of the body weight or body surface area of the animal. Yet, several other scaling methods have been proposed [25], based on, for example, the modeling of pharmacokinetic parameters where the variation of serum protein binding between species is taken into account.

Conclusions

The estimated human radiation burden of the 68Ge/68Ga generator eluate was slightly higher for females and similar for males as compared with somatostatin ligands 68Ga-DOTANOC,68Ga-DOTATOC, and 68Ga-DOTATATE, which is probably due to the retention in the liver and spleen. Our results revealed some differences between female and male rat data, which, at least in part, may be explained by the small sample size.

Notes

Declarations

Acknowledgements

The study was conducted within the Finnish Centre of Excellence in Cardiovascular and Metabolic Disease, supported by the Academy of Finland, University of Turku, Turku University Hospital and Åbo Akademi University. This study was sponsored by Eckert & Ziegler Radiopharma GmbH, Berlin, Germany, and further supported by a grant from the Academy of Finland (#258814). Helena Virtanen is a Ph.D. student financially supported by the Drug Research Doctoral Program, University of Turku Graduate School, Finland, and the Finnish Cultural Foundation. Aake Honkaniemi, Jussi Mäkilä and Jouni Tuisku are thanked for their help in PET imaging.

Authors’ Affiliations

(1)
Turku PET Centre, Turku University Hospital, University of Turku
(2)
Turku Center for Disease Modeling, University of Turku
(3)
Eckert & Ziegler Radiopharma GmbH
(4)
Preclinical Imaging and Drug Research, Turku PET Centre, University of Turku

References

  1. Fani M, Maecke HR. Radiopharmaceutical development of radiolabelled peptides. Eur J Nucl Med Mol Imaging. 2012;39:S11–30.View ArticlePubMedGoogle Scholar
  2. Maecke HR, Hofmann M, Haberkorn U. 68Ga-labeled peptides in tumor imaging. J Nucl Med. 2005;46 Suppl 1:172S–8.PubMedGoogle Scholar
  3. Meyer GJ, Mäcke H, Schuhmacher J, Knapp WH, Hofmann M. 68Ga-labelled DOTA-derivatised peptide ligands. Eur J Nucl Med Mol Imaging. 2004;8:1097–104.Google Scholar
  4. Roivainen A, Jalkanen S, Nanni C. Gallium-labelled peptides for imaging of inflammation. Eur J Nucl Med Mol Imaging. 2012;39:S68–77.View ArticlePubMedGoogle Scholar
  5. Uchino E, Tsuzuki T, Inoue K. The effects of age and sex on seven elements of Sprague-Dawley rat organs. Lab Anim. 1990;24:253–64.View ArticlePubMedGoogle Scholar
  6. Velikyan I, Xu H, Nair M, Hall H. Robust labeling and comparative preclinical characterization of DOTA-TOC and DOTA-TATE. Nucl Med Biol. 2012;39:628–39.Google Scholar
  7. Ujula T, Salomäki S, Autio A, Luoto P, Tolvanen T, Lehikoinen P, et al. 68Ga-chloride PET reveals human pancreatic adenocarcinoma xenografts in rats—comparison with FDG. Mol Imaging Biol. 2010;12:259–68.View ArticlePubMedGoogle Scholar
  8. Silvola JM, Laitinen I, Sipilä HJ, Laine VJ, Leppänen P, Ylä-Herttuala S, et al. Uptake of 68gallium in atherosclerotic plaques in LDLR−/−ApoB100/100 mice. EJNMMI Res. 2011;1:14.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Nanni C, Errani C, Boriani L, Fantini L, Ambrosini V, Boschi S, et al. 68Ga-citrate PET/CT for evaluating patients with infections of the bone: preliminary results. J Nucl Med. 2010;51:1932–6.View ArticlePubMedGoogle Scholar
  10. Pettinato C, Sarnelli A, Di Donna M, Civollani S, Nanni C, Montini G, et al. 68Ga-DOTANOC: biodistribution and dosimetry in patients affected by neuroendocrine tumors. Eur J Nucl Med Mol Imaging. 2008;35:72–9.View ArticlePubMedGoogle Scholar
  11. Hartmann H, Zöphel K, Freudenberg R, Oehme L, Andreeff M, Wunderlich G, et al. Radiation exposure of patients during 68Ga-DOTATOC PET/CT examinations. Nuklearmedizin. 2009;48:201–7.PubMedGoogle Scholar
  12. Sandström M, Velikyan I, Garske-Román U, Sörensen J, Eriksson B, Granberg D, et al. Comparative biodistribution and radiation dosimetry of 68Ga-DOTATOC and 68Ga-DOTATATE in patients with neuroendocrine tumors. J Nucl Med. 2013;54:1755–9.View ArticlePubMedGoogle Scholar
  13. Xavier C, Vaneycken I, D’huyvetter M, Heemskerk J, Keyaerts M, Vincke C, et al. Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 nanobodies for iPET imaging of HER2 receptor expression in cancer. J Nucl Med. 2013;54:776–84.View ArticlePubMedGoogle Scholar
  14. Roivainen A, Kähkönen E, Luoto P, Borkowski S, Hofmann B, Jambor I, et al. Plasma pharmacokinetics, whole-body distribution, metabolism, and radiation dosimetry of 68Ga bombesin antagonist BAY 86-7548 in healthy men. J Nucl Med. 2013;54:867–72.View ArticlePubMedGoogle Scholar
  15. International Commission on Radiological Protection. ICRP Publication 80. Recalculated dose data for 19 frequently used radiopharmaceuticals from ICRP publication 53. Ann ICRP. 1998;28:47–83.View ArticleGoogle Scholar
  16. Mird-dose estimate report no. 2. Summary of current radiation dose estimates to humans from 66Ga-, 68Ga-, and 72Ga-citrate. J Nucl Med. 1973;14:755–6.Google Scholar
  17. Parsey RV, Sokol LO, Bélanger MJ, Kumar JS, Simpson NR, Wang T, et al. Amyloid plaque imaging agent [C-11]-6-OH-BTA-1: biodistribution and radiation dosimetry in baboon. Nucl Med Commun. 2005;26:875–80.View ArticlePubMedGoogle Scholar
  18. Scheinin NM, Tolvanen TK, Wilson IA, Arponen EM, Någren KÅ, Rinne JO. Biodistribution and radiation dosimetry of the amyloid imaging agent 11C-PIB in humans. J Nucl Med. 2007;48:128–33.PubMedGoogle Scholar
  19. Santens P, De Vos F, Thierens H, Decoo D, Slegers G, Dierckx RA, et al. Biodistribution and dosimetry of carbon-11-methoxyprogabidic acid, a possible ligand for GABA-receptors in the brain. J Nucl Med. 1998;39:307–10.PubMedGoogle Scholar
  20. Harvey J, Firnau G, Garnett ES. Estimation of the radiation dose in man due to 6-18F-Fluoro-L-Dopa. J Nucl Med. 1985;26:931–5.PubMedGoogle Scholar
  21. Brown WD, Oakes TR, DeJesus OT, Taylor MD, Roberts AD, Nickles RJ, et al. Fluorine-18-fluoro-L-DOPA dosimetry with carbidopa pretreatment. J Nucl Med. 1998;39:1884–91.PubMedGoogle Scholar
  22. Tang G, Wang M, Tang X, Luo L, Gan M. Pharmacokinetics and radiation dosimetry estimation of O-(2-[18F]fluoroethyl)-L-tyrosine as oncologic PET tracer. Appl Radiat Isotopes. 2003;58:219–25.View ArticleGoogle Scholar
  23. Pauleit D, Floeth F, Herzog H, Hamacher K, Tellmann L, Müller HW, et al. Whole-body distribution and dosimetry of O-(2-[18F]fluoroethyl)-L-tyrosine. Eur J Nucl Med Mol Imaging. 2003;30:519–24.View ArticlePubMedGoogle Scholar
  24. Tolvanen T, Yli-Kerttula T, Ujula T, Autio A, Lehikoinen P, Minn H, et al. Biodistribution and radiation dosimetry of [11C]choline: a comparison between rat and human data. Eur J Nucl Med Mol Imaging.2010;37:874–83.View ArticlePubMedGoogle Scholar
  25. Leggett RW. Reliability of the ICRP’s dose coefficients for members of the public. 1. Sources of uncertainty in the biokinetic models. Radiat Prot Dosimetry. 2001;95:199–213.View ArticlePubMedGoogle Scholar

Copyright

© Autio et al. 2016

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.