Radioiodination and biological evaluation of ...

Radiochim. Acta 2016; 104(5): 345–353

Mahmoud Hamdi Sanad*, Mohamed Abdelmotelb Abelrahman, and Fawzy Mohamed Abdelmaged Marzook

Radioiodination and biological evaluation of levalbuterol as a new selective radiotracer: a 𝛽2-adrenoceptor agonist DOI 10.1515/ract-2015-2518 Received September 29, 2015; accepted December 2, 2015; published online January 22, 2016

Abstract: Levalbuterol was successfully radiolabeled with iodine using chloramine-T as an oxidizing agent via an electrophilic substitution reaction. The reaction parameters that affecting the labeling yield such as levalbuterol concentration, chloramine-T concentration, pH of the reaction medium and reaction time were studied in details. The radiochemical yield was 97.5 ± 0.5% and the radioiodinated compound was separated by HPLC. In vitro studies showed that the iodinated levalbuterol was stable for up to 24 h. The biodistribution in experimental animals showed that the lung uptake was 68.18 ± 0.17% at 5 min post injection which decreased with time until reached to 18.7 ± 0.12% at 2 h which was higher than other recent developed radiopharmaceuticals for lung imaging. The clearance pathways from the mice appear to proceed via both hepatobiliary and renal pathways. Predosing the mice with cold levalbuterol reduced the lung uptake to 20 ± 1.3% and further confirms the high specificity and selectivity of 125 I -levalbuterol for the lung. Keywords: Levalbuterol, radioiodination, imaging, lung,

𝛽2 -adrenoceptor.

Introduction Lung scanning is a test for evaluation of both pulmonary embolism and lung function before lung surgery and diagnosis of lung tumor [1–3]. Lung scanning techniques

*Corresponding author: Mahmoud Hamdi Sanad, Labeled Compound Department, Radioisotopes Production and Radioactive Sources Division, Hot Lab. Centre, Atomic Energy Authority, P.O. Box 13759, Cairo, Egypt, e-mail: [email protected] Mohamed Abdelmotelb Abelrahman, Fawzy Mohamed Abdelmaged Marzook: Labeled Compound Department, Radioisotopes Production and Radioactive Sources Division, Hot Lab. Centre, Atomic Energy Authority, P.O. Box 13759, Cairo, Egypt

were divided into two types where the first type is ventilation scan and the second type is perfusion scan. The two types of lung scan are usually done separately or together to evaluate the circulation of air and blood within a lungs [4–7].Ventilation scanning was performed to evaluate the movement of the air into and out of the bronchi and bronchioles where it can be done by inhalation of radioactive gases such as xenon or 99 Tc -DTPA in aerosols [8–14]. Lung perfusion scan used for measuring the pulmonary blood flow and allow detection of pulmonary embolism and lung cancer. Macro-aggregated albumin labeled with 99 Tc (99 Tc -MAA) has been reported as the most commercially used radiopharmaceutical for lung perfusion scanning [15–17] although it shows some limitations such as its particles size (∼ 30 microns) which may lead to particles trapping in the pre-capillary arterioles of the lungs after intravenous administration [18, 19]. Besides, it is derived from human serum albumin, which is collected from the pooled blood of human donors and so there is possibility of contamination by infective agents such as Variant Creutzfeldt Jakob disease, hepatitis B, hepatitis C and HIV [20]. While recombinant DNA technology is a new method for production of albumin to solve the drawbacks associated with human-derived HSA, but this method was relatively expensive and would increase the cost of routine imaging [21]. Therefore, there is urgent need to find a non-biological material (radiopharmaceutical products) to be used for lung perfusion agents without any limitations [22, 23]. Recently, many radiopharmaceuticals that is not sourced from human blood products such as 99 Tc(CO)5 I, 99 Tc -DHPM (99 Tc -5-etoxycarbonyl-4-phenyl6-methyl-3,4-dihydro-(1H)-pyrimidine-2-one) and 125/123 I IPMPD are used as potential lung perfusion agents showing maximum lung uptake of 21.4, 12.8 and 10.12% ID/g at 15, 60 and 2 min post injection, respectively [24–29]. Research continues for developing new radiopharmaceuticals of higher lung uptake and avoiding all disadvantages of 99 Tc -MAA to be used as better lung perfusion agents. The aim of the present work was to establish a simple and efficient method for radiosynthesis and biological evaluation of 125 I -levalbuterol. Factors affecting the radioiodina-

Authenticated | [email protected] author's copy Download Date | 5/5/16 8:35 PM

346 | M. H. Sanad et al., Radioiodination of levalbuterol / lung imaging / 𝛽2 -adrenoceptor

OH

OH

H

OH

H N

N HO

OH

Figure 1: Chemical structure of levalbuterol.

OH Na

125

OH

I

H N

CAT HO

HO 125

I

Figure 2: The proposed structure of radioiodinated levalbuterol.

tion process to obtain the maximum radiochemical yield were studied in details and the biodistribution studies of radioiodinated levalbuterol (Figure 1) were evaluated and examined as a new promising tool for lung perfusion imaging.

Materials and methods

the excess of iodine in order to stop the reaction and the radioiodinated product was isolated. Then, the radiochemical yield percent was determined using paper chromatography (PC) and paper electrophoresis and completely purified using HPLC column.

Reagents

Radiochemical analysis of 125 I-levalbuterol

No-carrier-added (NCA) sodium [125I ] iodide (3.7 GBq/ml) for radiolabelingwas purchased from Institute of Isotopes, Budapest, Hungary. Levalbuterol, Chloramine-T [Nchloro-p-toluene sulfonamide salt (CAT)] and methanol were purchased from Sigma-Aldrich. All chemicals were of analytical or clinical grade and were used directly without further purification unless otherwise stated. Deionized water was used in all experiments for the preparation of solutions, dilution and washing purposes.

Radiochemical yield of the 125 I -levalbuterol was determined by paper chromatographic method using strips of Whatman paper number 1. On paper sheet (1 cm width and 13 cm length), 1 – 2 μl of the reaction mixture was placed 2 cm above the lower edge and allowed to evaporate spontaneously. For development a fresh mixture of chloroform: ethanol (8.5 : 1.5 v/v) was used. After complete development, the paper sheet was removed, dried, and cut into strips, each strip is 1 cm width, and then each strip was counted in a well type 𝛾-counter. Radiochemical yield was further confirmed by paper electrophoresis. On Whatman paper sheet (2 cm width and 47 cm length), 1 – 2 μl of the reaction mixture was placed at 12 cm far from the cathode edge of the paper sheet. Electrophoresis is carried out for 1 h at voltage of 300 volt using normal saline (0.9% w/v NaCl solution) as electrolytes source solution. After complete development, the paper was removed, dried, and cut into strips, each strip is 1 cm width, and then the strip was counted in a well type 𝛾-counter. The percentage of radiochemical yield was calculated as the ratio of the radioactivity of 125 I -levalbuterol to the total activity multiplied by 100. HPLC analysis: The radiochemical yield of 125 I -levalbuterol was determined by direct injection of 20 μl of the reaction mixture at the optimum conditions for obtaining the highest radiochemical yields, into the column Lichrosorb RP-18 (250 mm × 4.6 mm, 5 μm) built in HPLC (Shimadzu model), which consists of pumps LC-9A, Rheohydron injector (Syringe Loading Sample Injector-7125) and UV spectrophotometer detector (SPD-6A) adjusted to the 276 nm wavelength using 50 mM ammonium bicarbonate (pH 7.8) (A) and acetonitrile (B) as a mobile phase. The flow rate was 0.8 ml/min and the fractions of 0.8 ml/min were collected separately us-

Radiolabeling procedure 125

I -levalbuterol (Figure 2) was generally synthesized by direct electrophilic radioiodination with NCA 125 I (𝑡1/2 = 60 d) in the presence of chloramine T (CAT) as oxidizing agent. Radioiodination conditions were investigated and optimized in order to maximize the radiochemical yield by using CAT (25 – 200 μg), concentration of levalbuterol (25 – 450 μg), room temperature, pH of the reaction (2– 11) and reaction time (5 – 60 min). Radiolabeling was carried out in a two-neck, 25 ml round bottomed flask fitted with a reflux condenser on one neck and the other neck was fitted with rubber stopper for withdrawing samples. The flask was immersed in a thermostatically controlled water bath. No-carrier-added Na125 I (7.2 MBq in 0.1% NaOH) was transferred to the reaction system and evaporated to dryness by vacuum. Accurately weighed 75 μg CAT was added to the reaction flask, then substrate (100 μg) was added that dissolved in water (1 ml). The reaction mixture was stirred with a magnetic stirrer and leave in room temperature for 15 min. Then 225 μg of (30 mg/ml) sodium metabisulphite (MBS) was added to decompose


Radichemical yield ,%

M. H. Sanad et al., Radioiodination of levalbuterol / lung imaging / 𝛽2 -adrenoceptor | 347

120 Application point 110 Radioiodination of Levalbuterol 100 90 80

Anode

70 60 50 40 30 20

ular weight of each one (distance from spotting point = 13 and 1 cm, respectively) as shown in Figure 3. Results of radiochemical yield from the two separation methods (paper chromatography and paper electrophoresis) are nearly the same. It was further confirmed by HPLC analysis [31], where the retention time of free iodide and 125 I levalbuterol were 4 and 9.8 min, respectively, and UV of levalbuterol was 9.61 min. as shown in the chromatogram (Figure 4).

Free Iodide

10 0 -4

-2

0

2

4

6

8

10

12

14

16

18

20

Distance,cm Figure 3: Electrophoresis radiochromatogram of 125 I-levalbuterol.

ing a fraction collector up to 20 ml and its activity was counted by using well a type NaI(Tl) crystal connected to a single-channel analyzer. The HPLC gradient was made up of (95% A) and (5% B) for the first 0 ∼ 5 min; a linear gradient of 80% A/20% B for 5 ∼ 10 min; a linear gradient of 50% A/50% B for 10 ∼ 15 min; a linear gradient of 20% A/80% B for 15 ∼ 17 min; a linear gradient of 5% A/95% B for 17 ∼ 19 min and a linear gradient of 95% A/5% B for 19 ∼ 20 min.

Factors affecting the percent labeling yield Effect of levalbuterol concentration The radiochemical yield as a function of levalbuterol concentration is depicted in Figure 5. The results indicated that the radiochemical yield increased from 80 to 97.5 ± 0.5% by increasing the amount of levalbuterol from 25 to 100 μg. Further increase in the amount oflevalbuterol beyond 100 μg had no impact on the labeling yield, which may be attributed to the fact that this amount of levalbuterol (100 μg) is enough to capture the entire generated iodonium ion as a result the yield reaches maximum value at this concentratio (97.5 ± 0.5%).

Biodistribution in normal mice Effect of chloramine-T (CAT) amount Organ distribution studies were carried out in a group of three male Albino Swiss mice. Each animal was injected in the tail vein with 0.2 mL solution containing 3.7 MBq of 125 I -levalbuterol. The mice were put in metabolic cages for the recommended time then anesthetized using chloroform. The organs as well as other body parts were dissected and fluids were collected carefully. Activity in each organ was counted and expressed as a percentage of the injected activity per organ. The weights of blood, bone and muscles were assumed to be 7, 10 and 40% of the total body weight, respectively [30].

Results and discussion The radiochemical purity of the 125 I -levalbuterol was determined using paper chromatography where radioiodide (I− ) remained near the origin (Rf = 0), while the 125 I -levalbuterol moved with the solvent front (Rf = 0.9). Radiochemical purity was further confirmed by paper electrophoresis where the free radioiodide and 125 I levalbuterol moved to different distances away from the spotting point towards the anode depending on the molec-

Chloramine-T is considered as an oxidizing agent that has the ability to oxidize the iodide (I− ) generating the highly reactive electrophilic species (H2 OI+ and HOI) which have important role in iodination reaction [32]. So the amount of chloramine-T is very important and critical in the iodination reactions. The data presented in Figure 6 clearly reveal that the radiochemical yield of 125 I -levalbuterol increased by increasing the amount of chloramine-T from 25 to 75 μg resulting in the highest radiochemical yield of 97.5 ± 0.5%. Increasing the CAT concentration above 75 μg leads to a decrease in the iodination yield where at 200 μg of CAT, the labeling yield was 60.33 ± 0.95%. This decrease in labeling yield may be attributed to the formation of undesirable oxidative byproducts like chlorination, polymerization and denaturation of levalbuterol [33]. The formation of these impurities may be attributed to the high reactivity and concentration of CAT [34]. Consequently, the optimum concentration of CAT (75 μg) is highly recommended in order to avoid the formation of by-products and to obtain high yield.



125 9.8 [ I-levalbuterol] % UV % Radioactivity

300000

Absorbance

Counts / sec

250000 200000 150000

9.61 [levalbuterol]

100000 50000

4 [Free Iodine]

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Retention time, min

Figure 4: HPLC chromatogram for 125 I-levalbuterol.

100

% Radiochemical yield

90 % Radioiodinated levalbuterol % Free Iodide

80 70 60 50 40 30 20 10 0 0

50

100

150 200 250 300 levalbuterol amounts,µg

350

400

450

Figure 5: Variation of the radiochemical yield of 125 I-levalbuterol as a function of different levalbuterol amounts; reaction conditions: 10 μl (∼ 3.7 MBq) Na125 I, 𝑥 μg levalbuterol, 75 μg of CAT, at pH 7, the reaction mixtures were kept at room temperature for 15 min.

100


90 80 70 60 % Radioiodinated levalbuterol % Free Iodide

50 40 30 20 10 0 0

25

50

75 100 125 150 Chloramin -T (CAT),µg

175

200

Figure 6: Variation of the radiochemical yield of 125 I-levalbuterol as a function of CAT; reaction conditions: 10 μl (∼ 3.7 MBq) Na125 I, 100 μg of levalbuterol, 𝑥 μg of CAT, at pH 7, the reaction mixtures were kept at room temperature for 15 min.



100



80 70 60 50 40 30

Figure 7: Variation of the radiochemical yield of 125 I-levalbuterol as a function of reaction time; reaction conditions: 10 μl (∼ 3.7 MBq) Na125 I, 100 μg of levalbuterol, 75 μg of CAT, at pH 7, the reaction mixtures were kept at room temperature for different intervals of time.

20 10 0 0

10

20

30 40 Reaction time,min

50

60

100


90 80 70 60

% Radioiodinated levalbuterol % Free Iodide

50 40 30 20 10 0 2

3

4

5

6

7

8

9

10

11

pH

Effect of reaction time The radiochemical yield of 125 I -levalbuterol was determined at different time intervals ranging from 5 to 60 min. The obtained results are illustrated in Figure 7. It is clear from the figure that the radiochemical yield is significantly increased when increasing the reaction time from 5 to 15 min. Increasing the reaction time to 60 min caused no significant change in the radiochemical yield. So, 15 min is the optimum reaction time required to attain maximum iodination.

Effect of pH The radiochemical yield of 125 I -levalbuterol complex was affected by changes in pH as graphically illustrated in Fig-

Figure 8: Variation of the radiochemical yield of 125 I-levalbuterol as a function of pH; reaction conditions: 10 μl (∼ 3.7 MBq) Na125 I, 100 μg of levalbuterol 75 μg of CAT, at different pH, the reaction mixtures were kept at room temperature for 15 min.

ure 8. Chloramine-T acts as an oxidizing agent in both acidic and alkaline media, the nature of active oxidizing species depends on the pH of the medium and the reaction condition. When dissolving chloramine-T in water, it decomposes to ArSO2 NCl, which undergoes hydrolysis in acidic medium to give HOCl. The hypohalous acid undergoes further hydrolysis to give H2 OCl+ . The possible oxidizing species in acidified CAT solutions are HOCl and H2 OCl+ and in alkaline solutions of CAT they are HOCl and ClO. The HOCl or H2 OCl+ generated oxidized the iodine under acidic conditions to the oxidative state I+ (iodonium) and thus rapidly reacts with any sites within levalbuterol that can undergo electrophilic substitution reactions [35, 36]. At pH 7, the yield was maximized (97.5 ± 0.5%) due to the great stability of levalbuterol structure at this pH value giving H+ , which was easily substituted by the active iodonium ion I+ . When the



100



80 70 60 50 40 30 20 10 0 10

20

30

40 50 Time post iodination ,h

60

pH of the reaction medium was shifted towards the acidic side, the yield decreased drastically reaching 55 ± 1.3% at pH 2. By shifting pH medium towards alkaline medium, the radiochemical yield decreased significantly reaching 55 ± 0.9% at pH 11. This may be attributed to the formation of hypoiodite ion (IO− ) and iodate (IO−3 ) ions which are not the suitable forms for radioiodination process [37, 38].

In-vitro stability of 125 I-levalbuterol The stability of 125 I -levalbuterol was studied in order to determine the suitable time for injection to avoid the formation of the undesired products that result from the radiolysis of the labeled compound. These undesired radioac-

70 Figure 9: In vitro stability of 125 I-levalbuterol.

tive products might be accumulated in non-target organs. Figure 9 clarifies the stability of 125 I -levalbuterol. The results show that 125 I -levalbuterol is stable for more than 70 h.

Biodistribution Table 1 shows the biodistribution of radioiodinated levalbuterol in important body organs and fluids. All radioactivity levels are expressed as average percent-injected dose per gram (% ID/g ± S.D). The in vivo instability of radioiodinated levalbuterol is commonly reflected by a high amount of radioactivity accumulation in the thyroid [39– 45]. So the low thyroid levels found at all the experi-

Table 1: Biodistribution of 125 I-levalbuterol in normal mice at different times. Organs and body fluids Blood Bone Muscle Brain Lungs Heart Liver Kidneys Spleen Intestine Stomach Urine Thyroid

5 15.2 ± 0.1 1.6 ± 0.10 1.3 ± 0.001 0.1 ± 0.001 68.18 ± 0.17 29.9 ± 0.21 2.6 ± 0.11 4.42 ± 0.17 2.0 ± 0.01 0.6 ± 0.01 0.91 ± 0.003 5.11 ± 0.21 0.9 ± 0.001

% I.D./g at different times post injection, min 15 30 60 10.0 ± 0.1 1.0 ± 0.003 1.2 ± 0.02 0.21 ± 0.002 60.4 ± 0.12 18.7 ± 0.82 4.9 ± 0.27 15.21 ± 0.21 1.30 ± 0.01 5.7 ± 0.39 0.40 ± 0.001 8.6 ± 0.11 0.8 ± 0.002

3.0 ± 0.11 1.95 ± 0.01 0.45 ± 0.003 0.11 ± 0.003 51.0 ± 0.15 9.3 ± 0.70 8.6 ± 0.50 24.0 ± 0.11 0.90 ± 0.001 9.2 ± 0.13 0.85 ± 0.001 13.1 ± 1. 0.9 ± 0.001

1.0 ± 0.001 1.88 ± 0.003 0.6 ± 0.001 0.10 ± 0.001 36.5 ± 0.13 8.1 ± 0.03 5.40 ± 0.71 38.2 ± 0.19 0.8 ± 0.002 11.8 ± 0.10 0.45 ± 0.001 25.0 ± 1.7 2.1 ± 0.01

120 0.9 ± 0.001 0.99 ± 0.002 0.06 ± 0.001 0.11 ± 0.002 18.7 ± 0.12 6.95 ± 0.66 3.85 ± 0.19 6.60 ± 0.7 0.30 ± 0.001 13.5 ± 0.16 0.7 ± 0.002 49.5 ± 0.63 2.9 ± 0.10

Mean ± SD (𝑛 = 5).


% Lung uptake,ID/g

M. H. Sanad et al., Radioiodination of levalbuterol / lung imaging / 𝛽2 -adrenoceptor |

75 70 Radioiodinated levalbuterol 65 60 55 50 45 40 35 30 25 20 15 10 5 0 0.00 0.25 0.50 0.75 Cold levosalbutomol amount ,µg

ment time points indicate that this radioiodinated levalbuterol was relatively stable in vivo [46–48]. The uptake within the kidneys were increased from 4.42 ± 0.17% at 5 min post injection to 38.2 ± 0.19% at 1 h post injection which decline to 6.60 ± 0.7% at 2 h post injection with increasing of urine uptake from 5.11 ± 0.21% at 5 min to 49.5 ± 0.63% at 2 h post injection. The high accumulation of the radioiodinated levalbuterol in lungs, 68.18 ± 0.17% at 5 min, is attributed to the fact that lungs function as reservoir for 𝛽2 receptor drugs. The activity in lung was decreased with time tell reached to 18.7 ± 0.12% at 2 h post injection which still higher than other recently discovered radiopharmaceuticals for lung imaging such as 99 Tc(CO)5 I, 99 Tc -DHPM (99 Tc -5-etoxycarbonyl-4-phenyl6-methyl-3,4-dihydro-(1H)-pyrimidine-2-one) and 125/123 I IPMPD are used as potential lung perfusion agents showing maximum lung uptake of 21.4, 12.8 and 10.12% ID/g at 15, 60 and 2 min post injection, respectively [49]. At 30 min post injection as in Table 1, it was concluded that this time was considered ideal time for lung imaging because the % of heart was 9.3 ± 0.70% and the % of blood was 3.0 ± 0.11% also the % of liver was 8.6 ± 0.50% but the % of lung was 51.0 ± 0.15 which considered more than the others. So lung/heart ratio equal to 5.48, lung/liver ratio equal to 5.93, and the lung/blood ratio equal to 17 at the same time post injection, so this time was more suitable for lung imaging.

Drug blocking study The blocking of 𝛽2 -receptor by the intravenous injection of cold levalbuterol result in extensive decrease in the accumulation of radioactivity of radiodinated leval-

1.00

351

Figure 10: Radioiodinated levalbuterol inhibition lung uptake in normal male Swiss Albino mice at 5 min p.i. (% ID/g ± SEM, 𝑛 = 5).

buterol within the lungs as it dropped from 68.18 ± 0.17 to 15.0 ± 0.8% ID/g by the injection of 1 μg of the cold substrate (Figure 10). These intensive decreases confirm the selectivity and high binding affinity of 125 I -levalbuterol to 𝛽2 -receptor located in lung.

Conclusions Electrophilic substitution reaction for levalbuterol with radioiodine in the presence of an in-situ oxidant such as chloramine-T is a convenient chemistry route for radiolabeling levalbuterol in high radiochemical yield (97.5%). This study described the in vitro and in vivo characterization of radioiodinated levalbuterol necessary for designing a potentially useful radiopharmaceutical for lung perfusion imaging. It showed both radiochemical and metabolic stability in vivo. Biological distribution studies in mice demonstrated the affinity and specificity of radioiodine levalbuterol for lung receptors.

References 1. PIOPED investigators, Value of the ventilation/perfusion scan in acute pulmonary embolism. Results of the prospective investigation of pulmonary embolism diagnosis (PIOPED), JAMA, 263(20): 2753 (1990). 2. Bajc M, Neilly JB, Miniati M, Schuemichen C, Meignan M, Jonson B, EANM guidelines for ventilation/perfusion scintigraphy. Part 1. Pulmonary imaging with ventilation/perfusion single photon emission tomography, Eur J Nucl Med Mol Imaging 36: 1356 (2009). 3. Parker JA, Coleman RE, Grady E, Royal HD, Siegel BA, Stabin MG, Sostman HD, Hilson AJ, SNM practice guideline for lung scintigraphy 4.0, J Nucl Med Technol 40(1): 57 (2012).



4. Hofman MS, Beauregard JM, Barber TW, Neels OC, Eu P, Hicks RJ, 68 Ga PET/CT ventilation-perfusion imaging for pulmonary embolism: a pilot study with comparison to conventional scintigraphy, J Nucl Med 52(10): 1513 (2011). 5. Akkas BE, Gokcora N, Atasever T, Yetkin I, The use of technetium-99m hexamethylpropylene amine oxime lung scintigraphy in the detection of subclinical lung injury in patients with noninsulin-dependent diabetes mellitus, J Nucl Med Commun 32(12): 1179 (2011). 6. Yang G, Wang X, Wang Z, Jiang Y, Fu J, Tc-99m MDP uptake in a giant pulmonary chondroma, J Clin Nucl Med 36(11): 1029 (2011). 7. Gu T, Shi H, Xiu Y, Gu Y, Primary pulmonary osteosarcoma: PET/CT and SPECT/CT findings, J Clin Nucl Med 36(12): e209 (2011). 8. Sakr TM, Synthesis and preliminary affinity testing of 123 I/125 IN-(3-iodophenyl)-2-methylpyrimidine-4,6-diamine as a novel potential lung scintigraphic agent, Radiochemistry 56(2): 170 (2014). 9. Mariano GD, Barbotte E, Basurko C, Comte F, Rossi M, Accuracy and precision of perfusion lung scintigraphy versus 133 Xeradiospirometry for preoperative pulmonary functional assessment of patients with lung cancer, Eur J Nucl Med Mol Imaging 33(9): 1048 (2006). 10. Nimmo MJ, Merrick MV, Millar AM, A comparison of the economics of xenon 127, xenon 133 and krypton 81m for routine ventilation imaging of the lungs, J Radiol 58(691): 635 (1985). 11. Rowe IF, Sleight PJ, Gaunt JI, Croft DN, Assessment of pulmonary ventilation scans using xenon-127 in the diagnosis of pulmonary embolis, Eur J Nucl Med 9(4): 154 (1984). 12. Stavngaard T, Sogaard LV, Mortensen J, Hanson LG, Schmiedeskamp J, Berthelsen AK, Dirksen A, Hyperpolarised 3 He MRI and 81𝑚 Kr SPECT in chronic obstructive pulmonary disease, Eur J Nucl Med Mol Imaging 32(4): 448 (2005). 13. Wu Y, Kotzer CJ, Makrogiannis S, Logan GA, Haley H, Barnette MS, Sarkar SK, A noninvasive [99𝑚 Tc]DTPA SPECT/CT imaging methodology as a measure of lung permeability in a guinea pig model of COPD, Mol Imaging Biol 13(5): 923 (2011). 14. Ogi S, Gotoh E, Uchiyama M, Fukuda K, Urashima M, Fukumitsu N, Influence of hilar deposition in the evaluation of the alveolar epithelial permeability on 99𝑚 Tc-DTPA aerosol inhaled scintigraphy, Jpn J Radiol 27(1): 20 (2009). 15. Sanad M, Novel radiochemical and biological characterization of 99𝑚 Tc-histamine as a model for brain imaging, J Anal Sci Technol 5(1): 23 (2014). 16. Zöphel K, Bacher-Stier C, Pinkert J, Kropp J, Ventilation/perfusion lung scintigraphy: what is still needed? A review considering technetium-99m-labeled macroaggregates of albumi, Ann Nucl Med 23: 1 (2009). 17. Sanad MH, Borai EH, Fouzy ASM, Chromatographic separation and utilization of labeled 99𝑚 Tc-valsartan for cardiac imaging, J Environ Sci Toxicol Food Technol 8(12): 10 (2014). 18. Miroslavova AE, Gorshkova NI, Lompov AL, Yalfimov AN, Suglobov DN, Ellis BL, Braddock R, Smith A, Prescott MC, Lawson RS, Sharma HL, Evaluation of 99𝑚 Tc(CO)5 I as a potential lung perfusion agent, J Nucl Med Bio 36(1): 73 (2009). 19. Seute T, Leffers P, ten Velde GP, Twijnstra A, Detection of brain metastases from small cell lung cancer: consequences of changing imaging techniques (CT versus MRI), Cancer 112(8): 1827 (2008).

20. El-Tawoosy M, Preparation and biological distribution of 99𝑚 Tccefazolin complex, a novel agent for detecting sites of infection, J Radioanal Nucl Chem 298: 1215 (2013). 21. Sanad MH, Synthesis and labeling of some organic compounds with technetium-99m, MSc thesis, Cairo (Egypt), Zagazig Univ. (Banha Branch), Faculty of Science (2004). 22. Perkins AC, Frier M, Bad blood and biologicals: the need for new radiopharmaceutical source materials, J Nucl Med Commun 20(1): 1 (1999). 23. Swidan MM, Sakr TM, Motaleb MA, El-Bary AA, El-Kolaly MT, Preliminary assessment of radioiodinated fenoterol and reproterol as potential scintigraphic agents for lung imaging, J Radioanal Nucl Chem 303: 531 (2015). 24. Sanad MH, Ibrahim IT, Radiodiagnosis of peptic ulcer with technetium-99m-pantoprazole, J Radiochemistry 55(3): 341 (2013). 25. Amin AM, Sanad MH, Abd-Elhaliem SM, Radiochemical and biological characterization of 99𝑚 Tc-piracetam for brain imaging, Radiochemistry 55(6): 624 (2013). 26. Abi-Dargham A, Zea-Ponce Y, Terriere D, Al-Tikriti M, Baldwin RM, Hoffer P, Charney D, Leysen JE, Laruelle M, Mertens J, Innis RB, Preclinical evaluation of [123I]R93274 as a SPECT radiotracer for imaging 5-HT2A receptors, Eur J Pharmacol 321(3): 285 (1997). 27. Sakr TM, Motaleb MA, Zaghary WA, Synthesis, radioiodination and in vivo evaluation of ethyl 1,4-dihydro-7-iodo-4oxoquinoline-3-carboxylate as a potential pulmonary perfusion scintigraphic radiopharmaceutical, J Radioanal Nucl Chem 303(1): 399 (2015). 28. EL-Ghany EA, Amin AM, EL-Sayed AS, EL-Kolaly MT, AbdelGelil F, Radiochemical and biological characteristics of 99𝑚 Tcpiroxicam for scintigraphy of inflammatory lesions, J Radioanal Nucl Chem 266(1): 125 (2005). 29. Motaleb MA, Amal SF, Ismail TI, Mona OS, Magda FI, Preperation and molecular molding of radioiodopropranolol as a novel potential radiopharmaceutical for lung perfusion scan, Int J Pharm Pharm Sci 7(8): 110 (2015). 30. Motaleb MA, El-Said H, Abdallah M, Atef M, Synthesis and evaluation of 99𝑚 Tc-NIDA and 99𝑚 Tc-DMIDA complexes for hepatobiliary imaging, J Radiochemistry 54(5): 501 (2012). 31. Ghemud AS, Santhakumari B, Pharne AB, Jadhav MM, Jain K, Kulkarni SM, Bioanalytical method development and validation of levalbuterol a 𝛽2 -adrenergic agonist by RP-HPLC method, Int J Pharm Pharm Sci 4(1): 249 (2012). 32. El-Kawy OA, Sanad MH, Marzook F, 99𝑚 Tc-Mesalamine as potential agent for diagnosis and monitoring of ulcerative colitis: labelling, characterisation and biological evaluation, J Radioanal Nucl Chem DOI:10.1007/s10967-015-4338-4 (2015). 33. Attila V, Sandor N, Zoltan K, Rezso GL, Rosch F, Handbook of Nuclear Chemistry, 2nd edn., Springer, New York (2011). 34. Saddar E, El-Tawoosy M, Motaleb HA, Preparation and biological evaluation of radioiodinated risperidone and lamotrigine as models for brain imaging agents, J Radioanal Nucl Chem 301: 189 (2014). 35. Abi-Dargham A, Zea-Ponce Y, Terriere D, Al-Tikriti M, Baldwin RM, Hoffer P, Charney D, Leysen JE, Laruelle M, Mertens J, Innis RB, Preclinical evaluation of [123 I] R93274 as a SPECT radiotracer for imaging 5-HT2A receptors, Eur J Pharmacol 321(3): 285 (1997).



36. Ramalingaiah H, Jagadeesh RV, Puttaswamy M, Os (VIII)catalyzed and uncatalyzed oxidation of biotin by chloramine-T in alkaline medium: comparative mechanistic aspects and kinetic modeling, J Mol Cat A: Chem 265(1–2): 70 (2007). 37. Abdel-Ghany IY, Moustafa KA, Abdel-Bary HM, Shams El-Din HA, Synthesis, radioiodination and biological evaluation of novel dipeptide attached to triazole-pyridine moiety, J Radioanal Nucl Chem 295: 1273 (2013). 38. Motaleb MA, Moustapha ME, Ibrahim IT, Synthesis and biological evaluation of 125 I-nebivolol as a potential cardioselective agent for imaging 𝛽1-adrenoceptors, J Radioanal Nucl Chem 289(1): 239 (2011). 39. Kuchar M, Oliveira MC, Gano L, Santos I, Kniess T, Radioiodinated sunitinib as a potential radiotracer for imaging angiogenesis-radiosynthesis and first radiopharmacological evaluation of 5-[125 I]Iodo-sunitinib, Bioorg Med Chem Lett 22(8): 2850 (2012). 40. Lee HY, Chung JK, Jeong JM, Lee DS, Kim DG, Jung HW, et al., Comparison of FDG-PET findings of brain metastasis from nonsmall-cell lung cancer and small-cell lung cancer, Ann Nucl Med 22(4): 281 (2008). 41. Taplin GV, Poe ND, A dualt lung-scanning technique for evaluation of pulmonary function, Radiology, 85: 365 (1965). 42. Decker RH, Wilson LD, Advances in radiotherapy for lung cancer, Semin Respir Crit Care Med 29(3): 285 (2008).

43. Liu HR, Zhao RR, Jiao XY, Wang YY, Fu M, Relationship of myocardial remodeling to the genesis of serum autoantibodies to cardiac 𝛽1-adrenoceptors and muscarinic type 2 acetylcholine receptors in rats, J Am Coll Cardiol 39(11): 1866 (2002). 44. De K, Chandra S, Sarkar B, Ganguly S, Misra M, Synthesis and biological evaluation of 99𝑚 Tc-DHPM complex: a potential new radiopharmaceutical for lung imaging studies, J Radioanal Nucl Chem 283: 621 (2010). 45. Abi-Dargham A, Zea-Ponce Y, Terriere D, Al-Tikriti M, Baldwin RM, Hoffer P, Charney D, Leysen JE, Laruelle M, Mertens J, Innis RB, Preclinical evaluation of [123 I]R93274 as a SPECT radiotracer for imaging 5-HT2A receptors, Eur J Pharmacol 321(3): 285 (1997). 46. Sanad, MH, Labeling and biological evaluation of 99𝑚 Tcazithromycin for infective inflammation diagnosis, J Radiochemistry 55(5): 539 (2013). 47. Sanad MH, Synthesis and labeling of some organic compounds with one of the mostradioactive isotope, Ph.D. thesis, Cairo (Egypt), Ain Shams Univ., Faculty of Science (2007). 48. Sanad MH, Ibrahim IT, Radiodiagnosis of peptic ulcer with technetium-99m labeled rabeprazole, J Radiochemistry 57(4): 435 (2015). 49. Sanad MH, Borai EH, Comparative biological evaluation between 99𝑚 Tc tricarbonyl and 99𝑚 Tc-Sn(II) levosalbutamol as a 𝛽2 -adrenoceptor agonist, J Radiochim Acta 103(12): 879 (2015).
