Preliminary dosimetric evaluation of 166Ho-TTHMP for human based on biodistribution data in rats

Applied Radiation and Isotopes 94 (2014) 260–265

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Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso

Preliminary dosimetric evaluation of on biodistribution data in rats

166

Ho-TTHMP for human based

Hassan Yousefnia a, Samaneh Zolghadri a,n, Amir Reza Jalilian a, Mojtaba Tajik b, Mohammad Ghannadi-Maragheh a a b

Nuclear Science and Technology Research Institute (NSTRI), Tehran 14395-836, Iran School of Physics, Damghan University, Damghan 36716-41167, Iran

H I G H L I G H T S


166Ho-TTHMP can be prepared in high radiochemical purity ( 4 99%, ITLC; 4 99.99%, HPLC).
The highest absorbed dose is observed in red marrow with 0.922 mSv/MBq.
All tissues receive insignificant absorbed dose in comparison with bone tissue.
166Ho-TTHMP has considerable characteristics compared to 166Ho-DOTMP.
166Ho-TTHMP can be a good candidate for bone marrow ablation.

art ic l e i nf o

a b s t r a c t

Article history: Received 13 November 2013 Received in revised form 11 August 2014 Accepted 31 August 2014 Available online 6 September 2014

In this work, the absorbed dose to each organ of human for 166Ho-TTHMP was evaluated based on biodistribution studies in rats by a RADAR method and was compared with 166Ho-DOTMP as the only clinically used Ho-166 bone marrow ablative agent. The highest absorbed dose for this complex is observed in red marrow with 0.922 mGy/MBq. The results show that 166Ho-TTHMP has considerable characteristics compared to 166Ho-DOTMP and can be a good candidate for bone marrow ablation in patients with multiple myeloma. & 2014 Elsevier Ltd. All rights reserved.

Keywords: 166 Ho-TTHMP Bone marrow ablation Internal dosimetry RADAR

1. Introduction Multiple myeloma is an aggressive plasma cell malignancy arising from plasma cells in the bone marrow with possibly fatal consequences (Child, 2003). Bone marrow ablation is a process whereby the human bone marrow cells are eliminated in preparation for a bone marrow transplant. This is performed using highintensity chemotherapy (Little and Storb, 2002) and total body irradiation (Moreau et al., 2002). It has been shown that the addition of skeletal targeted radiotherapy to the patients can improve the response rate in phase I and II trials, with promising long-term survival data (Giralt et al., 2003). Nowadays, many radiopharmaceuticals are developed for treatment of painful metastases. β  particles of low energies are

n

Corresponding author. Tel.: þ 98 21 88221103; fax: þ 98 21 88221107. E-mail address: [email protected] (S. Zolghadri).

http://dx.doi.org/10.1016/j.apradiso.2014.08.017 0969-8043/& 2014 Elsevier Ltd. All rights reserved.

recommended for bone pain palliation, whereas those with higher energies are used for bone marrow ablation (Bouchet et al., 2000). The most important point which should be considered in developing radiopharmaceuticals as bone pain palliative or bone marrow ablative agents is the absorbed dose delivered in bone marrow. 166 Ho with a half-life of 26.8 h and two principal β  -emissions (1.77 MeV [48%] and 1.85 MeV [51%] as the maximum beta energy) (Calhoun et al., 1992) is an excellent radionuclide for bone marrow ablation because the high energy of the radionuclide β  -emissions can give sufficient absorbed dose to the bone marrow. Also its physical half-life is short enough to permit delivery of high-dose chemotherapy and reinfusion of cryopreserved peripheral blood stem cells within 6–10 d (Breitz et al., 2006). Bone-seeking radiopharmaceuticals have been proposed for delivering ablative absorbed doses to marrow in multiple myeloma and other hematological malignancies. Various therapeutic possible 166Ho-labeled bone-seeking agents have been reported

H. Yousefnia et al. / Applied Radiation and Isotopes 94 (2014) 260–265

such as 166Ho-DOTMP (Breitz et al., 2006), 166Ho-EDTMP (Sohaib et al., 2011), 166Ho-APDDMP (Zeevaart et al., 2001), as well as 166 Dy/166Ho-EDTMP demonstrating adequate properties as a stable in vivo generator system for bone marrow ablation (Pedraza-López et al., 2004). The amount of energy deposited in unit mass of any organs by ionizing radiation, absorbed dose, plays an important role in evaluating the risks associated with the administration of radiopharmaceuticals and thus the maximum amount of activity that should be undertaken (Stabin et al., 1996). Many resources for facilitating dose calculations are available, once appropriate biokinetic data are gathered in animal or human experiments (Stabin, 2013). In nuclear medicine, the most commonly used method these days for calculation of the internal absorbed dose estimates is the radiation dose assessment resource (RADAR) method (Stabin and Siegel, 2003). Recently, 166Ho- triethylene tetramine hexa (methylene phosphonic acid) (166Ho-TTHMP) has been developed which showed significant uptake to the bone tissue (Zolghadri et al., 2013). In this work, the absorbed dose to each organ of human for 166Ho-TTHMP was evaluated based on biodistribution studies in rats by the RADAR method and was compared with 166Ho-DOTMP as the only clinically used 166Ho bone marrow ablative agent.

2. Materials and methods Natural holmium nitrate with purity of 499.99% was obtained from ISOTEC Inc. Production of 166Ho was performed using 165Ho (n, γ)166Ho nuclear reaction. Analytical HPLC to determine the specific activity was performed by Shimadzu LC-10AT, armed with two detector systems, a flow scintillation analyzer (Packard150 TR) and UV–visible (Shimadzu) using a Whatman Partisphere C-18 column 250  4.6 mm2 (Whatman Co., NJ, USA). A high purity germanium (HPGe) detector coupled with a Canberra™ (model GC1020-7500SL) multichannel analyzer and a dose calibrator ISOMED 1010 (Dresden, Germany) were used for counting distributed activity in rat organs. Calculations were based on the 80.6 keV peak for 166Ho. Animal studies were performed in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations, 2nd edn. Male healthy Wistar rats were purchased from Pasteur Institute, Tehran, Iran, and all weighing 180–200 g were acclimatized at proper rodent diet and 12 h/12 h day/night light/darkness. 2.1. Production and quality control of

166

HoCl3

In the first step, 100 mg of natural 165Ho(NO3)3 (165Ho, 99.99% from ISOTEC Inc.) was irradiated at a thermal neutron flux of 4  1013 n cm  2 s  1. The irradiated target was dissolved in 200 ml of 1.0 M HCl, to prepare 166HoCl3. The solution was filtered through a 0.22 mm filter (Millipore, Millex GV). The radionuclidic purity of the solution was checked using beta spectroscopy as well as HPGe spectroscopy. Also the radiochemical purity of the 166HoCl3 was studied using an instant thin layer chromatography (ITLC) method. 2.2. Preparation and quality control of 166

166

Ho-TTHMP

Ho-TTHMP was prepared according to the previously mentioned procedure (Zolghadri et al., 2013). Briefly, a stock solution of TTHMP was prepared by dissolution in 1 N NaOH and diluted to the appropriate volume with ultrapure water by dissolving 250 mg of TTHMP in 1.5 ml NaOH (2 N) and 3.5 ml distilled H2O, pH 12. Then 0.3 ml of this solution was added to 200 ml 166HoCl3 (211 MBq) (S.A. 47230.5 MBq/mg) and pH adjusted to 7 using phosphate buffer. The reaction mixtures were incubated with

261

stirring at room temperature for 1 h. The radiolabeling yield of the ligand was determined with paper chromatography using Whatman No. 2 paper in NH4OH:MeOH:H2O (2:20:40) mixture and High performance liquid chromatography (HPLC). 2.3. Biodistribution of

166

Ho-TTHMP in wild-type rats

Briefly, 200 μl of final 166Ho-TTHMP solution with 7.4 MBq radioactivity was injected intravenously into rats through their tail veins. The animals were sacrificed at 2, 4, 24 and 48 h postinjection. The activity concentration (A) of each tissue was calculated as (IAEA, 2004) A¼

N ε γ t s m k1 k2 k3 k4 k5

ð1Þ

where ε is the efficiency at photopeak energy, γ is the emission probability of the gamma line corresponding to the peak energy, t s is the live time of the sample spectrum collection in seconds, m is the mass (kg) of the measured sample, k1 , k2 , k3 , k4 and k5 are the correction factors for the nuclide decay from the time the sample was collected to the start of the measurement, the nuclide decay during counting period, self-attenuation in the measured sample, pulses loss due to random summing and the coincidence, respectively. N is the corrected net peak area of the corresponding photopeak given as N ¼ Ns

ts N tb b

ð2Þ

where N s is the net peak area in the sample spectrum, N b is the corresponding net peak area in the background spectrum and t b is the live time of the background spectrum collection in seconds. The ratio activity of different organs was calculated as percentage of injected activity per gram using the HPGe detector (%ID/g). For each time interval five animals were used. 2.4. Dosimetric studies The importance of an ideal bone marrow ablative radiopharmaceutical, especially with therapeutic applications, relays on the accumulation of the complex in bone compared to the critical organs such as the liver and kidneys since the radiation imposed to these organs as well as secondary irradiation to the neighboring organs is important in developing a therapeutic agent. Therefore, for such radiopharmaceuticals, clearance from blood, accumulation in bone and the ratio of accumulated activity in bone:critical organs are parameters which should be considered. The absorbed dose of each human organ was calculated by the RADAR method based on biodistribution data in Wistar rats. The accumulated activity in animals was extrapolated to the accumulated activity in humans by the proposed method of Sparks and Aydogan, (1996). OrganMasshuman =BodyMasshuman Α~ human organ ¼ Α~ animal organ OrganMassanimal =BodyMassanimal

ð3Þ

where à is the accumulated activity in the source organs and can be calculated by the following equation: Z 1 Α~ ¼ A ðtÞ dt ð4Þ t1

It should be noticed that A(t) is the activity of each organ at time t. The accumulated source activity for each organ of animals was calculated by plotting the percentage-injected activity versus time for each organ and computing the area under the curves. For this purpose the data points which represent the percentage-injected activity were created. An exponential curve was fitted to the last 3 points. These curves were continued to infinity and the integral

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of the area under the curves was calculated as the cumulative activity. Then the area under the curve was calculated and this accumulated activity was extrapolated to human according to the mean weights of each organ of human and rat given in Table 1 (Stabin and Siegel, 2003; Shanehsazzadeh et al., 2009)

Human organ weight (g)

Rat organ weight (g)

Bone Heart Stomach Kidneys Small intestine Spleen Muscle Liver Lungs Total body

6120 316 158 229 1100 183 28,000 1910 1000 73,700

1.90 0.65 0.99 1.47 3.80 0.68 101.53 8.13 1.24 190

DF ¼

166

HoCl3 (left) and

ð5Þ

k∑i ni Ei ϕi m

ð6Þ

where ni is the number of radiations with energy E emitted per nuclear transition, Ei is the energy per radiation (MeV), ϕi is the fraction of energy emitted that is absorbed in the target, m is the mass of target region (kg) and k is some proportionality constant (mGy kg=MBq s MeV). In this study DFs have been taken from the OLINDA/EXM software (Stabin et al., 2005).

Fig. 1. Gamma spectrum for

Fig. 2. ITLC chromatograms of

D ¼ N  DF

where N is the number of disintegrations that occur in a source organ (in Bq s=Bq) and DF (in mGy=MBq s) which represents the physical decay characteristics of the radionuclide, the range of the emitted radiations, and the organ size and configuration (Bevelacqua, 2005) is defined as

Table 1 The mean weights of organs of human and rat. Organ

The absorbed dose (D in mGy/MBq) is calculated by RADAR formulation Stabin and Siegel (2003)

166

166

HoCl3 solution used in the radiolabeling.

Ho-TTHMP solution (right) in NH4OH:MeOH:H2O (0.2:2:4) mixture.

H. Yousefnia et al. / Applied Radiation and Isotopes 94 (2014) 260–265

3. Results and discussion 3.1. Production and quality control of

166

HoCl3

The radionuclide was prepared in a research reactor with a range of specific activity between 3 and 5 GBq/mg for radiolabeling use. After counting the samples on the HPGe detector for 5 min, two major photons (5.4% of 80.68 keV and 0.9% of 1379.94 keV) were observed. Radionuclidic purity was higher than 99.96% (Fig. 1). 3.2. Preparation and quality control of

166

Ho-TTHMP

The radiolabled Ho complex was prepared in high radiochemical purity (499%, ITLC; 499.99%, HPLC). ITLC chromatograms of 166HoCl3 and 166Ho-TTHMP solution in NH4OH:MeOH:H2O (2:20:40) are shown in Fig. 2. HPLC chromatograms of 166HoTTHMP on a reversed phase column using acetonitrile:water 40:60 are presented in Fig. 3. 3.3. Biodistribution of

166

Ho-TTHMP in wild-type rats

The animals were sacrificed by CO2 asphyxiation at selected times after injection (2, 4, 24 and 48 h). Dissection began by drawing blood from the aorta followed by removing the heart, spleen, muscle, bone, kidneys, liver, intestine, stomach, lungs and skin samples. The tissue uptakes were calculated as the percentage of area under the curve of the related photopeak per g of tissue (% ID/g). The biodistribution values of 166Ho-TTHMP in different organs of Wistar rats are given in Table 2. Based on the results, it was concluded that the major portion of the injected activity of 166Ho-TTHMP was extracted from blood circulation into bones. Also due to the presence of anionic properties of the complex and relatively small size of the molecules, the complex is excreted through the kidneys. 3.4. Dosimetric studies Due to the direct relationship between absorbed dose and response in terms of cell killing/survival, calculation of the radiation absorbed dose to a targeted tissue is an important parameter which should be considered. The absorbed dose for each radiopharmaceutical can be developed by studying the biokinetics of the radiopharmaceutical in preclinical studies. In this study, dosimetric evaluation for 166Ho-TTHMP in human organs was

Fig. 3. HPLC chromatograms of

166

263

performed and compared with the absorbed dose in each human organ for 166Ho-DOTMP as the only clinically used 166Ho bone marrow ablative radiopharmaceutical. Similar to the methods used in absorbed dose estimation of the patients after injection of 166Ho-DOTMP, the accumulated source activity for each organ of rats after injection of 166Ho-TTHMP was calculated by plotting the injected activity (normalized by that of the total injected activity) versus time for each organ and computing the area under the curves. Finally, DF values have been taken from the OLINDA/EXM software. However, in this case the accumulated activity in the rat organs was converted to the accumulated activity in human organ by a mass extrapolation method. It is a common first step, consistent with the ICRP 62 recommendations (ICRP, 1993). However, extrapolation between animals and humans may lead to overestimation or underestimation of absorbed dose, but previous studies have demonstrated the usefulness of using animal biodistribution as a model for absorbed dose estimations in humans (Bélanger et al., 2008; Kesner et al., 2008). The clearance curves from each organ of the rats are shown in Fig. 4. The absorbed dose in each human organ after injection of 166 Ho-TTHMP is presented in Table 3. The data reported for the dosimetric assessments after 166Ho-DOTMP injection to the patients with multiple myeloma are also given in Table 3. As expected, the highest absorbed dose for 166Ho-TTHMP and 166HoDOTMP is observed in red marrow with 0.922 and 0.517 mGy/ MBq, respectively. This result shows that for a given absorbed dose to red marrow, lower activity of 166Ho-TTHMP needs to be injected. Table 2 Biodistribution values of Organ

Bone Liver Stomach Kidney Spleen Heart Blood Lung Intestine Muscle

166

Ho-TTHMP in different organs of Wistar rats.

%ID/g 2h

4h

24 h

48 h

1.96 0.28 0.12 1.13 0.14 0.10 0.21 0.14 0.12 0.02

1.57 0.18 0.06 0.57 0.26 0.09 0.31 0.17 0.05 0.08

1.5 0.08 0.03 0.53 0.06 0.02 0.03 0.06 0.04 0.03

0.55 0.02 0.03 0.30 0.04 0.01 0.01 0.05 0.04 0.01

Ho-TTHMP on a reversed phase column using acetonitrile:water 40:60, above: UV chromatogram, below: scintillation chromatogram.

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H. Yousefnia et al. / Applied Radiation and Isotopes 94 (2014) 260–265

Fig. 4. The clearance curves from each organ of the rats.

Table 3 The absorbed dose in each human organ after injection of

Tissue Adrenals Brain Breasts Gallbladder wall Lower large intestine wall Small intestine Stomach Upper large intestine wall

166

Ho-TTHMP and

166

Ho-DOTMP.

Absorbed dose (mGy/ MBq)166Ho-DOTMP

Absorbed dose (mGy/ MBq)166Ho-TTHMP

Tissue

Absorbed dose (mGy/ MBq)166Ho-DOTMP

Absorbed dose (mGy/ MBq)166Ho-TTHMP

0.014 0.014 0.013 0.013

0.002 0.003 0.001 0.001

Muscle Testes Pancreas Red marrow

0.013 0.013 0.013 0.517

0.016 0.001 0.001 0.922

0.014

0.045

Bone surfaces

0.920

1.14

0.013 0.013

0.001 0.011

Spleen Thymus

0.013 0.013

0.043 0.001

0.013

0.006

Thyroid

0.014

0.002

Heart wall

0.013

0.014

Kidneys Liver Lungs Reference

0.045 0.013 0.014 (Breitz et al., 2006)

0.127 0.019 0.041 This work

The effects of radionuclides in the management of disease are often estimated by the absorbed dose to target organ relative to normal tissues (Flynn et al., 2002). Therefore, red marrow to other tissue absorbed dose ratios for 166Ho-TTHMP and 166Ho-DOTMP has been compared in Table 4. While 166Ho-TTHMP demonstrated higher red marrow:total body and red marrow:liver uptake ratios than 166Ho-DOTMP, red marrow:kidney uptake ratio for 166HoDOTMP is higher than for 166Ho-TTHMP. According to this result, for a certain dose to the red marrow, total body and liver would receive lesser absorbed dose in the case of 166Ho-TTHMP rather than 166 Ho-DOTMP, while kidneys would receive higher absorbed dose.

Urinary bladder 0.291 wall Uterus 0.013 Total body 0.062 (Breitz et al., 2006)

0.001 0.001 0.099 This work

4. Conclusion 166 Ho-TTHMP was prepared in high radiochemical purity (499%, ITLC; 499.99%, HPLC). The final preparation was administered to wild-type rats and biodistribution of the complex was checked 2–48 h post-injection, showing major accumulation in the bone tissue. All tissues approximately receive insignificant absorbed dose in comparison with bone tissue (Table 4). The results show that 166Ho-TTHMP has considerable characteristics compared to the only clinically used bone marrow ablative radiopharmaceutical and therefore can be a good candidate for bone

H. Yousefnia et al. / Applied Radiation and Isotopes 94 (2014) 260–265

Table 4 Red marrow to other tissue absorbed dose ratios for

166

Ho-TTHMP and

166

Ho-DOTMP.

Tissue

166

166

Adrenals Brain Breasts Gallbladder wall Lower large intestine wall Small intestine Stomach Upper large intestine wall Heart wall Kidneys Liver Lungs Reference

36.9 36.9 39.8 39.8 36.9 39.8 39.8 39.8 39.8 11.5 39.8 36.9 ( Breitz et al., 2006)

379 337.7 1241.2 977.1 20.5 761.3 85.1 886.5 63.9 7.3 46.9 22.4 This work

Ho-DOTMP

265

Ho-TTHMP

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166

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39.8 39.8 39.8 1 0.6 39.8 39.8 36.9 1.8 39.8 8.3

56.2 1268.1 647.5 1 0.8 21.3 876.2 568.9 1201.3 958.3 9.3

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