Biodistribution and dosimetry of 177 Luoctreotate. evaluation of DMSA and lysine as kidney protecting agents in mice

Transcription

1 Biodistribution and dosimetry of 177 Luoctreotate and evaluation of DMSA and lysine as kidney protecting agents in mice Andreas Österlund M. Sc. Thesis 212 Supervisors: Emil Schüler Eva Forssell-Aronsson

2 Abstract 177 Lu- octreotate is used in treatments of patients with neuroendocrine tumours expressing somatostatin receptors (SSTR). To be able to optimize this treatment so more patients will undergo complete remission, more knowledge about the biodistribution is needed. The aims of this study was to study how the biodistribution of 177 Lu- octreotate in C57BL/6N mice depends on time after and quantity of injected amount, and to study the potential of DMSA as a kidney protector, alone and together with lysine, during treatment with 177 Lu- octreotate. C57BL/6N female mice were injected with 15 MBq 177 Lu- octreotate and killed 15 minutes, 3 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 3 days, 7 days and 14 days after, or injected with.1, 1, 5, 45, 9 and 15 MBq and killed 4 hours, 24 hours or 7 days after. Another group of mice were injected with 3.5 MBq 111 In- octreotide alone, or co- administrated with 1, 2, 4 or 8 mg DMSA, 4 or 8 mg lysine or with both DMSA and lysine, and killed 24 hours after. Radioactivity measurements were performed on blood, bone marrow, kidney, pancreas, spleen, lung and liver. Uptake as %IA/g and absorbed dose per unit injected activity was calculated. The results show, as expected, the highest absorbed dose in the kidneys. An uptake peak during the first hour, mainly due to the high activity concentration in the blood, was seen in lung, liver, pancreas and spleen. A second peak between 4 h and 8 h due to other uptake mechanisms such as binding to SSTR was seen. No limit for saturation of SSTR for 177 Lu- octreotate was found. The %IA/g uptake decreased when the amount of injected activity increased from.1 MBq for all studied organs except the kidneys. No clear relationship between amount of injected activity and kidney uptake was found, but of 4 mg DMSA seems to reduce the kidney uptake of 111 In- octreotide by 35 %. Injection with 8 mg lysine reduced the kidney uptake with 46 %, but when it was co- administrated with 2 mg DMSA no significant different from the control was seen, but there was a large variation in activity concentration in blood in these groups. DMSA may be used as kidney protector, but not when co- administered with lysine. More studies are needed to further elucidate kidney protection regimens. 2

3 Index Introduction... 4 Aims... 5 Materials and methods... 5 Animals... 5 Instruments... 5 Radiopharmaceuticals... 6 Calibration... 6 Administration and organ sampling... 7 Biodistribution of 177 Lu- octreotate with time... 7 Biodistribution of 177 Lu- octreotate effect of amount injected... 7 Biodistribution of 111 In- octreotide effect of kidney protection... 7 Measurement and measurement corrections... 8 Absorbed dose calculations... 8 Statistical analysis... 8 Results... 9 Calibration... 9 RCP control... 1 Biodistribution of 177 Lu- octreotate with time... 1 Biodistribution of 177 Lu- octreotate effect of amount injected Biodistribution of 111 In- octreotide effect of kidney protection... 2 Discussion Biodistribution of 177 Lu- octreotate Biodistribution of 111 In- octreotide effect of kidney protection Execution of the experiments Conclusion Acknowledgement References

4 Introduction The somatostatin analogue 177 Lu[DOTA,Tyr 3 ]octreotate is used for treatment of neuroendocrine (NE) tumours with overexpression of somatostatin receptors (SSTR) (1). 177 Lu is primarily a β- emitter, which makes it an effective radionuclide to use for therapy. The half- life of 177 Lu is 6.65 days and its daughter nuclide 177 Hf is stable (2). The energies of the beta particles are 149 kev(79%), 48 kev(12%) and 112 kev (9%). 177 Lu also has a γ- component, with energies of 28 kev(11%) and 113 kev (6%), which makes it possible to measure the activity with a γ- counter (2, 3). The mean electron range is.67 mm in water (3). Another clinically used β- emitter is 9 Y. The electron energy is much higher and the mean electron range is 12 mm in water (3). 9 Y is therefore less effective for treatments of small tumours, whereas 177 Lu is better suited for treatment of small tumours. Due to the fact that neuroendocrine tumours often have small metastases, 177 Lu is often the preferred choice (4). The treatments have shown good effects, but few patients undergo complete remission. Hitherto, the administrated activity is usually prescribed in a standard way (5), with the kidneys as the dose limiting organ. The initial dose limit to the kidneys of 23 Gy that was used was based on tolerance doses estimated for external beam radiotherapy (6), and now many clinics use a limit of 28 Gy. More knowledge about biodistribution, dosimetry and toxicity by internal radiation therapy is needed for better optimisation of this treatment modality. 111 In[DTPA,D- Phe 1 ]octreotide is used for scintigraphy of NE tumours expressing SSTR. The half- life of 111 In is 2.8 days, and it emits γ- radiation, 171 kev (91%) and 245 kev (94%). Its daughter 111 Cd is stable (7). There are five different subtypes of human somatostatin receptors (SSTR). Different NE tumour types express different amounts of the SSTR subtypes. Octreotate and octreotide have high affinity to SSTR2 and SSTR5, medium affinity to SSTR3 and low affinity to SSTR1 and SSTR4 (4). Some organs have high normal expression of SSTR, e.g. lung, pancreas and spleen(8), and with the lack of other uptake mechanisms, saturation effects after of 177 Lu- octreotate is likely to occur. Similar studies have not shown a level for saturation effects for different organs in mice (9). This is probably due to too few different injected activities. The accumulation of 177 Lu- octreotate in the kidneys is mainly due to the reabsorption process in the proximal tubular cells, but SSTR- mediated or peritubular uptake may play a role as well (1). Mouse kidneys have, like human kidneys, a relatively high expression of all somatostatin receptor subtypes (4). 177 Lu- octreotate is therefore retained in the tubular cells and may cause dose limiting nephrotoxicity (11). The reabsorption of peptides and proteins in the kidneys is due to several transport mechanisms: receptormediated endocytosis (megalin/cubulin receptors and somatostatin receptors), aminoacid/oligopeptide transporters, pinocytosis and passive diffusion (4). Megalin and cubulin are large endocytic receptors. Previous studies have shown that positively charged (basic) amino acids, e.g. arginine and lysine, reduce the kidney uptake by up to 4 % in humans, by blocking other ligands in the reabsorption process (1). Using 2,3-4

5 dimercaprosuccinic acid (DMSA), commonly used labelled with 99m Tc for renal scintigraphy, as a kidney protector has reduced the kidney uptake with 15,5 % 72 hours after in rats (12). No more studies are, to my knowledge, published, where DMSA is used as a kidney protector of 177 Lu- octreotate. Previous studies have investigated the biodistribution of 177 Lu- octreotate in mice, but these studies were performed on immune- deficient Balb/c mice transplanted with tumour tissue and only with at most 4 time points after investigated (3, 9, 13). Investigation of biodistribution of normal mice with retained immune system is thus of interest. Furthermore, it is presumed that the organ activity concentration reaches a maximum well before 24h after, why more detailed biodistribution studies are needed to elucidate the magnitude and time point of maximum uptake. Aims The aim of this study was to investigate the biodistribution of 177 Lu- octreotate in C57BL/6N mice. The study was divided into three experiments: 1) Biodistribution vs. time after. The aim of this experiment was to investigate how the biodistribution varies with time after, by studying the biodistribution at nine time points, from 15 minutes to 2 weeks after. 2) Biodistribution vs. amount of injected 177 Lu- octreotate. The aim of this experiment was to investigate how the biodistribution depends on the amount of injected 177 Lu- octreotate, and study potential saturation effects. Different amounts, from.1 to 15 MBq 177 Lu- octreotate, were injected, and biodistribution 4 h, 24 h and seven days after were studied. 3) Biodistribution vs. kidney protection agents. The aim of this study was to investigate how co- administration with DMSA and the positively charged amino acid lysine affecting the biodistribution of 111 In- octreotide. Materials and methods Animals Female C57BL/6N mice (Charles River, Salzfeld, Germany) with a weight of about 2 g were used. The mice were kept in groups of up to 8 individuals per cage. Food and water were given ad libitum. The experimental protocol was approved by the Ethics committee for Animal Research in Gothenburg. Instruments The activity in the tissue samples was measured with a Wizard 148 NaI(Tl) γ- counter, produced by Wallac, Finland, consisting of a 3 NaI(Tl) crystal, with pre- installed protocols for various radionuclides. The syringes (injected activities) were measured with a CRC- 15 dose calibrator ion chamber, produced by Capintec, IA, USA. The syringes were measured before and after, and the injected activity was calculate as the difference between these measurements, both decay corrected to the time of. 5

6 Radiopharmaceuticals 177 Lu- octreotate 177 LuCl 3 was acquired from I.D.B. Holland (I.D.B. Holland BV, Baarle-Nassau, the Netherlands). The activity of the delivered 177 LuCl3 was measured with the ionisation chamber and added to a quantity of octreotate calculated as!""!""!"!"#$%$#&!"!" (!"#)!"## (!"#) The solution was incubating at a temperature of 8 o C during 3 minutes. The solutions were cooled down for five minutes. A control of the radiochemical purity (RCP) was done with instant thin layer chromatography (ITLC). A volume of 1 μl 177 Lu- octreotate was diluted with 5 ml distilled water. Of this solution 1 μl were dropped on paper stripes with a width of 1.5 cm, 2 cm from the bottom of the stripe. The bottom of the stripe was placed in.1 M sodium citrate, ph 5.. When the liquid front reached 9 cm from the bottom of the stripe, the stripe was cut into two parts, 6 cm from the bottom. The 177 Lu activity at the bottom part and the top part were measured with the γ- counter. The RCP was calculated as RCP =!!"##"$!!!"!!"##"$!!!"#!!!!" %, where C!"##"$ is the number of counts in the bottom part, corresponding to 177 Lu- octreotate, C!"# the number of counts at the top part, corresponding to un- bound 177 Lu, and C!" the number of counts in a background measurement. Only solutions with a RCP value 99% was used. The RCP was also determined at 1, 2, 4 and 7 days after preparation, in order to investigate the degradation rate of the 177 Lu- octreotate. 111 In- octreotide 111 In- octreotide was bought from Covidien. The product name was Octreoscan. The labelling was done according to the producer, and diluted to desired concentration. Calibration The sensitivity of the γ- counter was determined in relation to the ionisation chamber. An original solution of 3.98 MBq 177 Lu- octreotate measured with the ionisation chamber, was used. The solution was then diluted. Samples of μl with different activity concentrations were measured with the γ- counter. A calibration curve to translate the number of counts into activity was made. The total activity in each sample was 1, 5, 1, 2, 5, and 321 kbq. Due to death time effects, two approximations were made, one for CPS count- rates < 31 and one for count- rates > 31. This limit (31) is the CPS- value of the intersections between the two lines. A calibration curve was also made for 111 In. A linear approximation was used for solutions with activities of 1, 5 and 1 kbq. For higher activities the death time correction was needed (death time factor>1.2).. 6

7 For both radionuclides, 1 MBq diluted into different volumes between 5 and 2 μl was measured, for studying effects of measurement geometry. For 111 In, 1 MBq diluted into μl were places in Eppendorf tubes before placement in the scintillation vial. Administration and organ sampling The mice were i.v. injected in the tail vein. The animals were killed by cardiac puncture under anaesthesia (Pentobarbitalnatrium vet. APL 6 mg/ml, APL, Umeå, Sweden). Samples of blood, lung, liver, pancreas, spleen, kidney and bone marrow were collected, weighed and placed in a 2 ml scintillation vial. The bone marrow samples were first placed in an Eppendorf tube before the tube was placed in the scintillation vial. In the first two experiments, a volume of 1 μl blood was also taken and the concentrations of red blood cells, white blood cells and platelets were measured (SYSMEX POSH i, Kobe, Japan). Biodistribution of 177 Lu- octreotate with time The 177 Lu- octreotate solution was diluted with a NaCl- solution until an activity concentration of MBq/ml. Totally, 36 mice were divided into nine groups with four mice in each group. Each mouse was injected with about 15 MBq 177 Lu- octreotate. The activities in the syringes were measured with the ionisation chamber before and after. The groups were killed at 15 minutes, 3, minutes, 1 h, 4 h, 8 h, 24 h, 3 days, 7 days and 14 days after. Biodistribution of 177 Lu- octreotate effect of amount injected In order to investigate the dependence of quantity of injected activity, three time points were chosen (4h, 24 h and 7 days after ). For each of these time points, six groups with four mice each were used. The groups were injected with.1, 1, 5, 45, 9 and 15 MBq 177 Lu- octreotate in a volume of.15 ml. Data from mice injected with 15 MBq were taken from the first experiment, described above. A control group only injected with NaCl- solution was killed 24 h after. The concentration of blood cells in the two studies described above was compared with this group. Biodistribution of 111 In- octreotide effect of kidney protection In this study the radiopharmaceutical 111 In- octreotide, the amino acid lysine and dimercaptosuccinic acid (DMSA) were used. Each mouse was injected with 3.5 MBq 111 In- octreotide in a volume of.1 ml. Some mice were also injected with 1, 2 or 4 mg DMSA in a volume of.5 ml, or 8 mg in a volume of.1 ml. The syringes with 4 mg or 8 mg lysine contained a volume of.5 ml each. Each group consisted of 4 mice and all mice were killed 24 h after of 111 In- octreotide. DMSA was injected at time, or 1 h or 2 h before of 111 In- octreotide. For each of the three times 1, 2, 4 or 8 mg DMSA were injected. One group was injected with 4 mg and another group with 8 mg lysine at the same time as of the 111 In- octreotide- solution. 7

8 Another four groups were injected with 4 mg lysine at the same time as the 111 In- octreotide- solution, one of them together with of 1 mg DMSA, one together with 2 mg DMSA, one where 1 mg DMSA was injected 1 hour before the of the lysine and 111 In- octreotide, and one where 2 mg DMSA where injected 1 hour before the of the lysine and 111 In- octreotide. Two groups were injected with 8 mg lysine, 2 mg DMSA and 111 In- octreotide, one where all three syringes where given at the same time, one where DMSA where given one hour before the of 111 In- octreotide and lysine. One control group was injected with 111 In- octreotide only. Measurement and measurement corrections The activities in the organs were measured with the gamma counter using the calibration curve. The measurement time was between 3 seconds and 3 minutes for each organ. If the number of counts were at least above the background level and the death time factor smaller than 1.2 the measurement was accepted. The number of counts were multiplied with a death time correction factor, and divided by the measurement time in seconds, and corrected for background. This value was translated into activity (kbq) using the calibration curves. The activity was decay corrected back to the time of. The injected activity in each mouse was calculated by subtracting the rest activity from the activity in the syringe before, when all activities were decay corrected to the time of. The measured activity was divided by the injected activity and also by the mass of the organ. This was multiplied by to get the unit %IA/g (per cent of injected activity per gram tissue) for the activity concentration. Absorbed dose calculations To estimate the mean absorbed dose per injected activity in the organs the MIRD formalism was used. The mean absorbed dose D was calculated as D = A! (n!e! ) φ m where A is the cumulated activity during the time period of interest,!(n! E! ) is the mean energy emitted by nuclear transformation, ϕ is the absorbed fraction and m the organ mass.!(n! E! ) was 147 kev for β, ce, and auger radiations from 177 Lu (2) and ϕ was set to 1 (electrons). Ã was determined by drawing straight lines between the points in an activity vs time plot, and calculate the area under the curve. This was done for the first 2 weeks after, using nine points in the study of biodistribution over time, and during the first week after in the study of dependence of quantity of injected activity. These two different ways of estimating the cumulated activity were also compared for 15 MBq injected activity. The mean absorbed dose was divided by the amount of injected activity, and displayed as Gy/MBq. Statistical analysis A plot viewing the mean activity concentration over time for each organ was made. The, SEM! value for each point was calculated as SEM! =!!!! 8!! (!!!!)!!!!,

9 where n is the number of samples, x is the mean value for the samples and x! the value for each sample. In some figures the ratio R between different mean uptake values x! and x! are shown. The SEM! value was calculated as SEM! = R!"#!!!!! +!"#!!!!! (14). Results Calibration The results of the calibration measurements for 177 Lu are presented in figure 1. Also included are the linear regression curves, for activities lower and higher than 1 kbq respectively. Those are used for estimating the activity in the samples. calibration Lu y =.576x R² = Activity (kbq) y =.36x R² = CPS Figure 1 Results of the calibration measurement for 177 Lu. Two different calibration curves for 177 Lu, activities lower than 1 kbq and higher than 1 kbq is shown. From these results following expressions are derived: A =.36 CPS CPS 31 A =.576 CPS 8.28 CPS > 31 where A is the activity in kbq, and CPS the background- corrected counts per second (CPS) measured with the γ- counter. Figure 2 shows the calibration results for 111 In. 9

10 Calibration In- 111 up to 1 kbq (deathtime factor <1.2) 12 kbq y =.43x R² = CPS Figure 2 Results of the calibration measurement for 111 In, up to activities of 1 kbq. From these results the following expression for the 111 In activity is derived: A =.43 CPS. No volume dependence was found during measurement with the γ- counter, neither for 177 Lu nor 111 In. For 111 In no difference was found if the activity was placed in an Eppendorf tube before it was put into the scintillation vial. RCP control Table 1 shows the results of the RCP- control. The radiochemical purity was higher than 99% day, 1 and 2, where day is the day when the preparation was done. Table 1 Radiochemical purity of 177 Lu- octreotate. Day is the day when the preparation was done Day RCP mean SEM 99.8%.1% %.% %.2% %.3% %.4% Biodistribution of 177 Lu- octreotate with time In the following figures each data point represents the mean value of four samples. The error bars represent ±SEM value, n=4. The activity concentration of 177 Lu- octreotate in the bone marrow, blood, liver, lungs, spleen and pancreas during the first 2 weeks after is shown in Figure 3, and for kidneys in Figure 4. For liver, pancreas, lung and spleen, a first uptake peak is seen during the first hour, and a second peak between four and eight hours. The blood concentration decreased from 9.3 %IA/g at 15 minutes to.58 %IA/g at 1 hour after 1

11 , and shows after 1 hour no more significant peak. For kidneys, the concentration decreased from 77 %IA/g at 15 minutes to 11 %IA/g at 8 hours after. After 8 h the amount of activity still decreased but with a lower speed. For bone marrow a peak was found after 24 h, although large individual differences between bone marrow uptake in this group was seen, why the SEM- value is relatively high. 11

12 14 Biodistribution 15 min- 14 d after 12 %IA/g min 3 min 1h 4h 8h 24h 3d 7d 14d bone marrow Blood Liver Lung Spleen Pancreas Biodistribution 8 h- 14 d after 1,6 1,4 1,2 %IA/g 1,8,6,4 8h 24h 3d 7d 14d,2 bone marrow Blood Liver Lung Spleen Pancreas Figure 3 Uptake during the first 2 weeks after in bone marrow, blood, liver, lung, spleen and pancreas. Each bar represents the mean value of 4 samples ±SEM. Uptake from 8h- 14 days after are displayed in an own chart. Note that the values for bone marrow 24 h after and pancreas 8h after are higher than the range of the scale, and is better seen in the first chart. 12

13 %IA/g Biodistribution kidney 15 min- 14 d after Kidney 15 min 3 min 1h 4h 8h 24h 3d 7d 14d %IA/g Biodistribution kidney 8h- 14d after Kidney 8h 24h 3d 7d 14d Figure 4 Uptake during the first 2 weeks after in kidney. Each point represents the mean value of 4 samples ±SEM. Uptake from 8h- 14 days after are displayed in an own chart. Figure 5 shows the percentage ratio between the concentration of red blood cells (RBC), white blood cells (WBC), haemoglobin (HGB) and platelets (PLT) and the concentration in the control group for different time- points after. The variations of the concentration of white blood cells were large, but no clear relationship of variation over time was seen. For platelets, red blood cells and haemoglobin no large differences from the control group was seen. 13

14 % of control group 15 5 PLT h 4h 8h 24h 3d 7d 14d min min Time after % of control group WBC h 4h 8h 24h 3d 7d 14d min min Time after RBC HGB % of control group h 4h 8h 24h 3d 7d 14d min min Time after % of control group min 3 min 1h 4h 8h 24h 3d 7d 14d Time after Figure 5. Percent of concentration of red blood cells (RBC), white blood cells (WBC), haemoglobin (HGB) and platelets (PLT) for different time- points after, compared to the concentration in a control group only injected with NaCl- solution. Each bar represents the mean value of 4 samples ±SEM Figure 6 shows the activity concentration (%IA/g) in bone marrow, liver, lung, spleen, kidney and pancreas, divided by the activity concentration (%IA/g) in blood for the same time point. During the first hour, these ratios seems to be approximately constant for liver and spleen, slowly increasing for lung and faster increasing for pancreas, blood marrow and kidneys. 14

15 Tissue/Blood activity concentrations (%IA/g in tissue)/(%ia/g in blood) 1 1 bone marrow Liver Lung Spleen Kidney Pancreas,1,1 1 1 Time (h) Figure 6 Activity concentration (%IA/g) in bone marrow, liver, lung, spleen, kidney and pancreas, divided by the activity concentration (%IA/g) in blood for different times after. Each point represents the mean value of 4 samples +SEM. The x and y axes have logarithmic scales. Estimation of absorbed dose per unit injected activity during the first two weeks after is shown in Table 2. Table 2 Estimation of absorbed dose per injected activity (Gy/MBq) at different times after in studied organs. The mice were injected with 15 MBq 177 Lu- octreotate. [Gy/MBq] Bone Blood Liver Lung Spleen Kidney Pancreas marrow 15 min min h h h h d d d Biodistribution of 177 Lu- octreotate effect of amount injected The following figures shows how the 177 Lu concentration described as %IA/g, depends on amount of injected 177 Lu- octreotate. The results 4 h, 24 h and 7 d after are shown in each figure. The x- and y- axes has a logarithmic scale. Figure 7 shows the results for liver, Figure 8 for the lungs, Figure 9 for pancreas, Figure 1 for the kidneys, 15

16 Figure 11 for spleen, Figure 12 for bone marrow, and Figure 13 for blood. For lungs, it seems like %IA/g depends on the amount of injected activity following a potential function (R 2 >.97 for 4 h, 24 h and 7 d after, respectively). For the other organs except liver and kidney, no such clear similar relationships were found. However, the tendency is that %IA/g decreases when the amount of injected activity increases, for all studied organs except kidney. 1 Liver- saturation effects %IA/g 1,1 4 h after 24 h after 7 d after,1,1 1 1 Injected ativity (MBq) Figure 7 Concentration (%IA/g) in liver for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 days after. Each data point represents the mean value of 4 samples+sem. The x and y- axes have logarithmic scales. 16

17 Lungs - saturation effects %IA/g 1 1,1,1,1 1 1 Injected activity (MBq) 4 h after 24 h after 7 d after %IA/g = *IA R² =.9716 (4h) %IA/g = 4.535*IA R² = (24h) %IA/g =.922*IA R² = (7d) Figure 8 Concentration (%IA/g) in lung for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 days after. Each data point represents the mean value of 4 samples+sem. The x and y- axes have logarithmic scales. %IA/g 1 1,1 Pancreas- saturation effects 4 h after 24 h after 7 d after %IA/g = *IA R² = (4h) %IA/g=.8324*IA R² = (24h),1,1 1 1 Injected activities (MBq) %IA/g =.1876*IA R² = (7d) Figure 9 Concentration (%IA/g) in pancreas for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 days after. Each data point represents the mean value of 4 samples+sem. The x and y- axes have logarithmic scales. 17

18 Kidney - saturation effects %IA/g 1 4 h after 24 h after 7 d after 1,1,1 1 1 Injected activity (MBq) Figure 1 Concentration (%IA/g) in kidney for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 days after. Each data point represents the mean value of 4 samples+sem. The x and y- axes have logarithmic scales. %IA/g Spleen- saturation effects 1 1,1,1,1 1 1 Injected activity (MBq) 4 h after 24 h after 7 d after %IA/g= *IA -,489 R² =.9225 (4h) %IA/g=.3645*IA -,324 R² = (24h) %IA/g=.1126*IA -,163 R² = (7d) Figure 11 Concentration (%IA/g) in spleen for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 days after. Each data point represents the mean value of 4 samples+sem. The x and y- axes have logarithmic scales. 18

19 %IA/g Bone marrow 1 1,1,1,1 1 1 Injected activity (MBq) 4 h after 24 h after 7 d after %IA/g= *IA -,842 R² = (4h) %IA/g=.5652*IA -,5 R² =.61 (24h) %IA/g=.444*IA -,55 R² = (7d) Figure 12 Concentration (%IA/g) in bine marrow for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 days after. Each data point represents the mean value of 4 samples+sem. The x and y- axes have logarithmic scales. %IA/g Blood - saturation effects 1,1,1,1,1,1 1 1 Injected activyty (MBq) 4 h after 24 h after 7 d after %IA/g =.241*IA -,414 R² = (4h) %IA/g =.291*IA -,222 R² =.2868 (24h) %IA/g=.65*IA -,45 R² = (7d) Figure 13 Concentration (%IA/g) in blood for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 days after. Each data point represents the mean value of 4 samples+sem. The x and y- axes have logarithmic scales. The concentration of red blood cells (RBC), white blood cells (WBC), haemoglobin (HGB) and platelets (PLT) for different amounts of injected 177 Lu- octreotate, at 4 h, 24 h and 7 days after, respectively, is shown in Figure 14. The variation in concentration of white blood cells was largest, and in most of the groups the concentration of all blood cells were higher than in the control group. 19

20 Figure 14 The concentration of red blood cells (RBC), white blood cells (WBC), haemoglobin (HGB) and platelets (PLT) for different amounts of injected 177 Lu- octreotate, 4 h, 24 h and 7 d after. Each bar represents the mean value of 4 samples±sem. Estimation of absorbed dose during the first week after of different amounts of 177 Lu- octreotate is shown in Table 3. Table 3 Estimation of mean absorbed dose per injected activity (Gy/MBq) in organs for the first week after of 177 Lu- octreotate. Injected activity (MBq) Pancreas Kidney Spleen Lung Liver blood Bone marrow Biodistribution of 111 In- octreotide effect of kidney protection Figure 15 shows the dependence of kidney uptake due to amount injected DMSA. A decreasing of kidney uptake with 35%±17% and 34%±15% was seen when 4 mg DMSA was injected respectively 1h before the of 111 In- octreotide. A decreasing with 2

21 36%±19% was seen when 1 mg DMSA was injected 2h before of the activity, and an increasing by 44%±28% when 8 mg DMSA was injected at the same time as the activity. Kidney uptake depending of DMSA % of uptake in control group mg DMSA h - 1h - 2h Figure 15 % of kidney uptake in control group depending on injected amount DMSA and time for relative to of 111 In- octreotide. Each bar represents the mean value of 4 samples ±SEM. Figure 16 shows the kidney uptake due to amount injected lysine. The uptake was 84.6%± 2.4 % of uptake in control group for 4 mg injected lysine and 54.9%±21. % of uptake in control group for 8 mg injected lysine. 12 Kidney uptake lysine + In % of uptake in control group mg Lysin 4 mg Lysin Figure 16 % of kidney uptake in control group depending on injected amount lysine. Each bar represents the mean value of 4 samples ±SEM 21

22 Figure 17 shows the kidney uptake then both DMSA and Lysine were injected. No significant difference from control is seen. Kidney uptake DMSA+lysine+In % of uptake in control group mg dmsa 4 mg lysine 2 mg dmsa 8 mg lysine 2 mg dmsa 4 mg lysine h - 1h Figure 17 % of kidney uptake in control group depending on different amount DMSA and lysin, and diffrent times for DMSA relative to of 111 In- octreotide. Each bar represents the mean value of 4 samples ±SEM. Figure 18 shows the organ uptake for different times and different amounts of DMSA. 22

23 Organ uptake DMSA injected at time h % of uptake in control group mg DMSA (h) 2 mg DMSA (h) 4 mg DMSA (h) 8 mg DMSA (h) blood bone marrow kidney liver lung pancreas spleen Organ uptake DMSA injected at time - 1h % of uptake in control group mg DMSA (- 1h) 2 mg DMSA (- 1h) 4 mg DMSA (- 1h) 8 mg DMSA (- 1h) blood bone marrow kidney liver lung pancreas spleen % of uptake in control group Organ uptake DMSA injected at time - 2h 5 1 mg DMSA (- 2h) 2 mg DMSA (- 2h) 4 mg DMSA (- 2h) 8 mg DMSA (- 2h) blood bone marrow kidney liver lung pancreas spleen Figure 18 % of organ uptake in control group depending on different amount DMSA injected at, 1 and 2 h before of 111 In- octreotide. Each bar represents the mean value of 4 samples ±SEM 23

24 Figure 19 shows the organ uptake for different amount Lysine and DMSA injected and different times of the DMSA relative to the 111 In- octreotide s. 25 Organ uptake % of uptake in control group blood bone marrow kidney liver lung pancreas 8 mg Lysin 4 mg Lysin 1 mg dmsa 4 mg lysine (h) 1 mg dmsa 4 mg lysine (- 1h) 2 mg dmsa 8 mg lysine (h) 2 mg dmsa 8 mg lysine (- 1h) 2 mg dmsa 4 mg lysine (h) 2 mg dmsa 4 mg lysine (- 1h) spleen Figure 19 % of organ uptake in control group depending on different amount DMSA and lysine, DMSA injected at different times relative to 111 In- octreotide. Each bar represents the mean value of 4 samples ±SEM. Discussion In this study, the biodistribution of 177 Lu- octreotate in mice was investigated. A further study where the effects of DMSA and lysine were also conducted, to investigate the effects on the biodistribution following of these kidney protectors. Hopefully, the studies will be important to further optimize treatments of NE tumours with 177 Lu- octreotate. Biodistribution of 177 Lu- octreotate The study of the biodistribution of 177 Lu- octreotate with time showed that the highest uptake value for blood, kidneys, spleen, lungs and liver was found at the first time- point measured (15 min after ). Pancreas showed a significant uptake peak during the first hour. These organs, except the kidneys, also showed a local minimum value at 1 h after. A probable reason for this early peak is the high amount of blood in these organs together with a high activity concentration in blood early after. The activity concentration in blood decreased fast from 15 min to 1 h after, which is mainly due to excretion via the kidneys. Furthermore, in liver and spleen, the tissue- to- blood concentration ratios were approximately constant, between.3 and.5, during the first hour. The conclusion of this is that the activity measured in liver and spleen during the first hour mainly is located in the blood, and that blood represents 3-5% of the organ masses. In the lungs and pancreas, the corresponding tissue- to- blood activity concentration ratios increased fast to values above 1. An explanation for these high 24

25 values is that 177 Lu- octreotate is actively taken up by these tissues during the first hour. Despite the fast decrease in activity concentration in the organs, dosimetric estimations showed that 6-9 % of the absorbed dose in lung, spleen, pancreas and liver given during the first two weeks, was absorbed during the first hour, why this peak would be interesting to investigate further. The bone marrow is next to the kidneys a potentially dose limiting organ. Usually, according to the MIRD scheme, the accumulated bone marrow activity is calculated as the accumulated activity in the blood. In this study, the bone marrow was removed from the femoral bone and the activity concentration was determined. The results show, in the first two experiments, that the blood and bone marrow activity concentration curves had a similar shape but the uptake (%IA/g) in the bone marrow was consistently higher, with the exception for the first hour after in the study where biodistribution with time was studied. However, the uncertainty, represented by the SEM value, for bone marrow was relatively high. The reason for this could be uncertainties in determination of the very small masses that were extracted from the bone (up to 4 mg), and the difficulty to remove only pure bone marrow All organs, except the kidneys, showed a decreasing uptake when the injected activity increased from.1 to 1 MBq, where lungs showed the largest relative reduction. However, it is important to note that the absolute uptake still increased with the amount of injected 177 Lu- octreotate. This indicates that one part of the uptake depends on the amount of injected 177 Lu- octreotate, and one doesn t. Previous studies have shown limits for saturation in lung and spleen by of between.1 and 1 μg 111 In- octreotide in tumour- bearing nude mice, and for lower amounts the uptake (%IA/g) increased with the amount of injected activity(15). Since DOTA- octreotate has a higher affinity for SSTR than DTPA- octreotide (16) a lower limit for saturation was expected. In this study the amounts of injected octreotate were between.4 and 6 μg, and it was expected to find a level for saturation in these organs in our mouse model. Such a level was not found, when the uptake concentration (%IA/g) started to decrease. This may indicate that the SSTR receptors partially are saturated even at very low amounts, or differences between the SSTR expressions between the species. Mouse kidneys have a high expression of all five SSTR subtypes (17, 18), however no saturation effects were seen. The reason for that is probably that other uptake mechanisms such as endocytosis via megalin/cubulin receptors, amino acid/oligopeptide transporters, pinocytosis or passive diffusion dominate (4). Rather an increasing of the uptake (%IA/g) when the injected activity increases is seen, except for the highest amounts, where the results deviate after 4 h and 24 h after. By comparing this study with similar studies by Dalmo et al.(9) and Schmitt et al.(3), where tumour- bearing nude mice were injected and studied 1 day and 7 days after, our results show approximately the double activity concentration for liver, pancreas and spleen for the same amount injected activity, with the exception for pancreas 24h after in the study by Dalmo et al.(9). In a study by de Araújo et.al.(13), where,74 MBq 177 Lu- octreotate was injected in tumour- bearing nude mice, approximately the same activity concentration was found for liver, pancreas, and spleen as in this study. The most prominent difference is that the kidneys and lungs in the present study show uptake levels (%IA/g) 2-5 times higher for kidneys and 3-1 times higher for lung. The calculated absorbed dose is about the double in kidneys and the 25

26 absorbed dose in pancreas is also at least twice as high as in Dalmo et al.(9). The reason for the higher uptake in kidney is probably due to that the tumour bearing mice have a high uptake in the tumour, but in our model this extra activity is mainly excreted through the kidneys. Variances between the mouse strains may be a reason for the differences as well. From the results, it is hard to see a relationship between the concentration of blood cells and time after or quantity of injected activity. Consistently, most of the groups show a larger blood cell concentration than the control group for all types of blood cells. If the SEM- value multiplies with 1.96, a 95 % interval for the percentage ratio is seen. Many of the groups injected with activity, especially in the study where dependence of injected activities was studied, the ratio is significantly higher than % with 95% confidence. No relationship between the amount of 177 Lu- octreotate or time, just that injected 177 Lu- octreotate often leads to increased blood cell concentration. The variation of white blood cells is largest, and often one of the samples in a group departs from the other ones, why the SEM- value is high. These results are not as expected. Due to the fact that blood cells are produced in the bone marrow, high uptake in the bone marrow probably can affect the production of blood cells, due to radiation damages of the bone marrow cells. It was proposed that a change could be seen for high activities, but the effects would be larger if it was studied longer times after. The expected effect was a lower concentration of blood cells, not a higher. According to this, these results are probably due to large individual differences between individual mice. In the control group no big individual differences is shown, and it also shown a small SEM value. The conclusion is that in groups where some individuals shows a naturally higher blood cell concentration, it will easily be higher than the control group, where these variations is not seen. Even high doses one week after shows a higher concentration. One week is probably too short to see the expected effects. Biodistribution of 111 In- octreotide effect of kidney protection Basic amino acids, such as lysine, are commonly used as a radio protector during treatment with 177 Lu- octreotate. The aim of this experiment was to investigate the more unexplored kidney protector DMSA, and also combine it with the more explored kidney protector lysine, to get better knowledge about how DMSA block the kidneys from octreotate or octreotide. If other uptake ways were blocked than for lysine, a larger decrease would be seen by combining the two peptides. DMSA was originally developed for reducing neurotoxicity from toxic heavy metals, and is mostly absorbed in the upper part in the loop of Henle in the proximal tubular cells in the kidneys(12). Previous studies have shown a decreased uptake of about 4% flowing co- of lysine in rat kidney, as opposed to of 111 In- octreotide alone (1). The same dosage in our mouse model corresponds 8 mg lysine per mouse. The significant decrease with 45 % (95 % confidence) that is seen in our study when 8 mg lysine is injected is as expected. The results for when using co- of DMSA shows that the largest reduction of kidney uptake is seen when 1 mg DMSA was injected 2 h before the 111 In- octreotide, and when 4 mg is injected at the same time or 1h before the of the activity. All groups where DMSA was injected 1 h or 2 h before the activity, have a lower mean value than the control group just injected with 111 In- octreotide. Moorin et al.(12) saw in a 26

27 preliminary study an increased uptake of 43 % in rat kidney when.15 mg/g DTPA was injected i.p. (intra peritoneal) 1 h before of 111 In- octreotide. This was a small study with just two animals in each group, so the uncertainties are large. The amount.15 mg/g corresponds 3 mg/mouse in our model. The time for of DMSA cannot easily be compared between these studies, because in this study the DMSA was injected directly into the blood stream. However, no prominent increase is seen for either 2 mg or 4 mg of injected DMSA, independent of time for. The results from Moorin et al.(12) cannot be confirmed with this study. The authors discuss that a possible reason for the increasing uptake could be an indication of that DMSA remove Lu 3+ from 177 Lu- octreotate and it will be bound to the DMSA. Moorin et al.(12) also saw a decreased uptake of 8,5% after 24 h and 15,5% after 72 h if DMSA was injected i.p..5 h after of 111 In- octreotide, because DMSA then was not able to remove Lu 3+. In our study, DMSA was injected prior or at the same time as of 111 In- octreotide. While it doesn t seems to be any uptake of 111 In on the DMSA, no disadvantage with of DMSA prior 111 In- octreotide may be seen. From our results it is hard to see a dependent of time for at all. In summary, of 4 mg DMSA seems to decrease the kidney uptake, independent of time for. An increase in kidney uptake when 8 mg DMSA was injected at the same time as the activity is hard to explain, because similar effects are not seen by 1h and 2h before of the activity. The low uptake when 1 mg was injected 2h before of 111 In- octreotide is also hard to explain. Except of one group DMSA seems to have a positive effect when quantities from 4 mg and above were injected. No tendencies for changes in other organs when DMSA was injected were seen. This was similar to the previous study(12). Interesting results is shown when both DMSA and lysine was injected. No significant difference from the control group was seen. It seems like of DMSA or lysine alone reduce the kidney uptake more effectively than a combination. High mean values and SEM- values for concentration in blood and bone marrow was also found in these groups.. Execution of the experiments Of the 196 mice injected in this study, 23 i.v. s failed. Regarding the seven mice in seven different groups in the kidney protection study, the of either 111 In- octreotide, lysine or DMSA failed in such a way that the injected amount was impossible to quantify. These mice were removed from the study and the data were not included in the analysis. The other 16 s were subcutaneous, but the mice were kept in the study, since previous studies have shown that the uptake of 111 In- octreotide 24 h after is similar, independent of if the activity was injected intravenously or subcutaneously(15). However, in the kidney protection study, the subcutaneous would lead to a slower uptake of the substance in the blood, potentially affects the uptake in the kidney, where time for the substances reaching the kidney may be important. Such relationship was not found in this data. In the determination of the activity concentrations in samples, no correction for sample volume was done. Experimentally, no volume dependence was found during measurement with the γ- counter, neither for 177 Lu nor 111 In. For 111 In no difference was found if the activity was placed in an Eppendorf tube before it was put into the 27

28 scintillation vial. The conclusion was that for these small organ samples no corrections for self- absorption need to be done. All organs were placed in the bottom of the scintillation vial. The bone marrow was placed in an Eppendorf tube after extraction, due to difficulties in placing the bone marrow in the bottom of the scintillation vial. When the Eppendorf tube was placed in the scintillation vial, the bone marrow sample was in the bottom part of the vial and the measurement geometry was similar to the other tissue samples. Conclusion 1) Biodistribution vs. time after. A peak in liver, pancreas, lung and spleen during the first hour after, mostly due to the high blood concentration, was found. A second lower peak between 4 and 8 hours was found in these organs. As expected the highest absorbed dose per unit injected activity was found in the kidneys. The activity concentration in bone marrow is higher but seems to follow the activity concentration in the blood. 2) Biodistribution vs. amount of injected 177 Lu- octreotate. Because tendencies could be studied, individual variances will play a minor role when dependence of amount of injected activities was studied. A decreasing of %IA/g is seen even for small amounts of injected activities (from.1 MBq), in blood, bone marrow, liver, pancreas, spleen and lung. %IA/g in kidney is relatively independent of amount injected 177 Lu- octreotate between 1 and 45 MBq. No limit for saturation of somatostatin receptors was found. 3) Biodistribution vs. kidney protection agents. 4 mg DMSA seems to reduce the kidney uptake of 111 In- octreotde with about 35%, but co- with lysine shows no protecting effect. It would be interesting to further develop the protecting effects when 177 Lu- octreotate is used, and confirm increased uptake of 177 Lu if DMSA is injected prior 177 Lu- octreotate. 28

29 Acknowledgement I want to thank my supervisors, Emil Schüler and Eva Forssell- Aronsson, for their engagement, feedback and support during this work. I also want to thank Ann Wikström and Lilian Karlsson for their skilled performance with the animals, Johan Spetz for helping me with the γ- counters, and Maria Larsson for her expertise and assistance during the kidney protection project. Without you this work would be nothing. References 1. Kwekkeboom DJ, Kam BL, van Essen M, Teunissen JJ, van Eijck CH, Valkema R, et al. Somatostatin- receptor- based imaging and therapy of gastroenteropancreatic neuroendocrine tumors. Endocrine- related cancer. 21;17(1):R Epub 29/12/1. 2. National nuclear Data Center. Medical Internal Radiation Dose 23; Available from: 3. Schmitt A, Bernhardt P, Nilsson O, Ahlman H, Kolby L, Schmitt J, et al. Biodistribution and dosimetry of 177Lu- labeled [DOTA,Tyr3]octreotate in male nude mice with human small cell lung cancer. Cancer biotherapy & radiopharmaceuticals. 23;18(4): Epub 23/9/ Forssell- Aronsson E, Spetz J, Ahlman H. Radionuclide Therapy via SSTR: future aspects from experimental animal studies. Neuro- endocrinology Kwekkeboom DJ, Teunissen JJ, Bakker WH, Kooij PP, de Herder WW, Feelders RA, et al. Radiolabeled somatostatin analog [177Lu- DOTA,Tyr3]octreotate in patients with endocrine gastroenteropancreatic tumors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 25;23(12): Epub 25/4/2. 6. Emami B, Lyman J, Brown A, Coia L, Goitein M, Munzenrider JE, et al. Tolerance of normal tissue to therapeutic irradiation. International journal of radiation oncology, biology, physics. 1991;21(1): Epub 1991/5/ National Nuclear Data Center. Medical Internal Radiation Dose. 29; Available from: 8. Uhlen M, Bjorling E, Agaton C, Szigyarto CA, Amini B, Andersen E, et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Molecular & cellular proteomics : MCP. 25;4(12): Epub 25/8/3. 9. Dalmo J, Rudqvist N, Spetz J, Laverman P, Nilsson O, Ahlman H, et al. Biodistribution of 177Lu- octreotate and 111In- minigastrin in female. Oncology reports. 212;27(1): Epub 211/1/ Rolleman EJ, Melis M, Valkema R, Boerman OC, Krenning EP, de Jong M. Kidney protection during peptide receptor radionuclide therapy with somatostatin analogues. European journal of nuclear medicine and molecular imaging. 21;37(5): Epub 29/11/ Vegt E, de Jong M, Wetzels JF, Masereeuw R, Melis M, Oyen WJ, et al. Renal toxicity of radiolabeled peptides and antibody fragments: mechanisms, impact on radionuclide therapy, and strategies for prevention. Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 21;51(7): Epub 21/6/18. 29

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