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PREPARATION AND EVALUATION

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PREPARATION AND EVALUATION PREPARATION AND EVALUATION Document Transcript

  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 PREPARATION AND EVALUATION OF 99m Tc-UBIQUICIDIN 29-41 FOR INFECTION IMAGING 1 Chalermsin Permtermsin1 , Nipavan Poramatikul2 and Sudkanung Phumkhem2 1. Office of Atoms for Peace, 16 Vibhavadee-Rangsit Road, Chatuchak, Bangkok, Thailand 10900 2. Thailand Institute of Nuclear Technology, 16 Vibhavadee-Rangsit Road, Chatuchak, Bangkok, Thailand 10900 ABSTRACT In this study Tc-99m labeled ubiquicidin 29-41 peptides (UBI 29-41) were prepared into three formulations. Formulation 1, 2 and 3 was 5, 25 and 50 µg of UBI 29-41 labeled with 4-6 mCi Tc-99m, respectively. The radiochemical purity at various conditions and biodistribution of Tc-UBI 29-41 in bacteria infected mice were evaluated. The radioactivity recovery and radiochemical purity for 99mTc-UBI 29-41 was > 80 % determined by reverse phase HPLC and Sep-Pak C-18 cartridge. The stability of three formulations of 99mTc-UBI 29-41 after diluted with saline for 1-6 h showed the radiochemical purity > 70 % and stability in serum at 37 0C showed > 60 % determined by Sep-Pak C18 cartridge. The dissociation of formulation 1 in cysteine solution showed indifferently. The dissociation of formulation 2 and 3, after 6 h of incubation with 500 molar ratio of cysteine were 21.94 ± 5.50 and 34.89 ±7.96 %, respectively. The biodistribution of 99mTc-UBI 29-41 showed a fast renal clearance and liver displayed gradual decline in uptake values with formulations 3 < 2 < 1. The accumulation of 99m Tc-UBI 29-41 in the thigh muscle of mice infected with S. aureus showed T/NT ratio as 0.9-4.9 and biodistribution of 99mTc-UBI 29-41 in K. pneumoniae infected mice exhibited T/NT ratio as 1.3-9.1. These data indicated that labeled peptide preferentially tag microorganism at the site of infection. KEYWORDS:: infection, biodistribution, technetium, UBI 1. INTRODUCTION On the basis of medical history, physical examination, imaging studies, and laboratory testes, the clinician determines whether signs and symptoms in patients are suggestive of an infectious or non-infectious cause. Moreover, in patients with serious underlying conditions, the identification of infection and initiation of treatment at early stages of the disease are critical for a favorable outcome. At the start of the antibacterial treatment, microbiologic cultures are performed to identify the infectious agent and its in vitro susceptibility to antibiotics. The latter results may or may not correlate with clinical outcome. In addition, the prescription of antibiotics based on pharmacodynamic principles that predict bacterial eradication and prevent the emergence of resistance is important to the success of therapy [1]. In view of the need to reduce resistance emergence and to minimize costs, the ability to monitor the efficacy of antimicrobial agents can be important asset. In this connection, scintigraphic quantification of the accumulation of technetium-99m labeled with ubiquicidin (UBI) at the site of infection may be an option. 99mTc labeled UBI 29-41 is a radiolabeled synthetic fragment of ubiquicidin that preferentially binds to bacteria and fungi over mammalian cells [2]. In support of this suggestion, it was found that this radiolabeled peptide distinguished bacterial and fungal infections from sterile inflammatory processes [3,4,5,6,7]. More over, a significant correlation between accumulation of 99mTc-UBI 29-41 and the number of viable bacteria at the site of infection was noted [3]. Based on these considerations, the aim of this study was to formulate and investigate 99mTc-UBI 29-41 in infected mice. 1 Corresponding author. Tel: 0-2579-5230 ext. 5912, 5913 Fax: 0-2562-0125 E-mail: chalermsinp@yahoo.com
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 2. MATERIALS AND METHODS 2.1 Materials Peptide based on cationic, TGRAKRRMQYNRR (1,693 Da), 29-41 of α-helical domains of human ubiquicidin (UBI 29-41) was a gift of IAEA. Peptide was diluted to stock concentrations of 1 mg/ml in injectable water, aliquoted into 50 µl/microtube and stored at –20 0C before used. Tin (II) chloride dihydrate (Merck); L- Cysteine (Sigma); Trifluoro acetic acid (TFA, Sigma) Tryptic Soy Agar (Difco); Tryptic Soy Broth (Difco); Technetium generator (Biogenetech Cis Bio International); Sep-Pak C-18 cartridge (Waters, Milford, MA); ITLC-SG (Gelman Sciences); other chemicals were analytical grade. 2.2 Methods 2.2.1 Direct labeling of UBI peptide with technetium-99m UBI 29-41 was directly labeled with technetium-99m as described earlier [3]. Briefly, 5-50 µg of peptide solution was added to a 1.5 ml microtube containing 5 µl of a fresh 1 mg/ml solution of stannous chloride in 10 mM HCl. To this was added 10-15 µl of 0.1 N NaOH, followed by 4-6 mCi 99mTc-pertechnetate in a volume of 100-400 µl and the volume was adjusted to 500 µl with 0.9 % normal saline (final pH 8.8-11.5). The labeled solution was incubated at room temperature for 5 min and the pH was adjusted to 6.8-7.5 with 0.1 N HCl or 0.1 N NaOH. This preparation was referred to here as 99mTc-UBI 29-41. In this study, 3 formulations were formulated: 4-6 mCi 99mTc-pertechnetate was added in 5, 25 and 50 µg of UBI 29-41, these refers to formulations 1, 2 and 3, respectively. 2.2.2 Quality control Radiochemical purity analysis was performed by Sep-Pak C-18 cartridge open column chromatography, instant thin layer chromatography (ITLC) and reverse phase high-performance liquid chromatography (HPLC). The radiochemical purity was performed by Sep-Pak C-18 mini cartridge (Waters, Milford, MA) preconditioned with 5 ml of ethanol followed by 5 ml of 1 mM HCl. An aliquot of 20-100 µl of the labeled peptide was loaded on the cartridge and eluted with 5-10 ml of 1 mM HCl to elute 99mTc-pertechnetate. The labeled peptide was elute with 5-10 ml of ethanol:saline (1:1) followed by 5-10 ml of 0.1 M HCl:methanol (15:85). The hydrolyzed-reduced 99mTc remained in the column. ITLC silica gel glass fiber strips (Gelman Sciences) were used as stationary phase. Saline solution or methyl ethyl ketone was the mobile phase to determine the amount of free 99mTcO4- (Rf=1). Reverse phase HPLC method was carried out with a Waters instrument (Waters, USA) on a C-18 column (Jupitor, Phenomenex) with both radioactivity and UV spectrophotometer in-line detectors. The gradient was run at a flow rate of 1 ml/min using the following conditions: 0.1% trifluoroacetic acid (TFA)/water (solvent A) and 0.1% TFA/acetonitrile (solvent B). The gradient started with 100% solvent A for 3 min, changed to 50% solvent A over 10 min, was held for 10 min, changed to 30% solvent A over 3 min and finally returned to 100% solvent A over 4 min. Recovery of the radioactivity was routinely determined. 2.2.3 Serum stability An equal volume of labeled peptide solution and human serum was incubated at 37 0C for 1, 2 and 6 h. The amounts of free pertechnetate in the samples were determined by ITLC using methyl ethyl ketone as eluent and the radiochemical purity was performed by Sep-Pak C-18 mini cartridge as previously described. 2.2.4 Dilution stability To determine the stability of 99mTc-UBI 29-41 after dilution in saline solution, radiolabeled peptide was diluted to a ratio 1:20 with 0.9 % normal saline at pH 7 and 9. The reaction mixture was analyzed by Sep-Pak C-18 and ITLC. 23
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 2.2.5 Cysteine challenge The 99mTc-UBI 29-41 was tested for instability toward cysteine. A fresh cysteine solution was prepared (10 mg/ml in sterile water) and diluted to different concentrations. Then, the radiolabeled was incubated with cysteine to a molar ratio of 1:5 and 1:500. Each tube was incubated at 37 0C and radiochemical purity was analyzed at 1, 2 and 6 h by Sep-Pak C-18 mini cartridge. 2.2.6 Biodistribution in normal mice Male Swiss Albino Mice weight range 20-30 g (4-8 weeks) were purchased from National Laboratory Animal Centre, Mahidol, Thailand. The animals were housed in the animal housing facilities of The Radioisotope Production Program, Office of Atoms for Peace. Food and water were given ad libitum. Preparation containing radiolabeled peptide was diluted in 0.9 % normal saline and then injected into a tail vein. After injection of 99mTc-UBI 29-41 (50 µCi/mouse), accumulation of radioactivity in various organs was determined. The various organs were dissected, weighed and counted for radioactivity at the various time intervals. Data are expressed as the percentage of total 99mTc-activity of the injected dose/gram of tissue (% ID/ g). 2.2.7 Biodistribution in bacterial infected mice K. pneumoniae and S. aureus, approximately 1 X 107 colony forming unit in 0.1 ml saline solution were injected into the right thigh muscle of each mouse. After 24 h, preparation containing radiolabeled peptide was diluted in saline and then injected into a tail vein (50 µCi/0.1 ml/mouse). At 1, 2 and 4 h after injection, accumulation of radioactivity in various organs was determined as previously described. 2.2.8 Statistical analysis All experiments were evaluated with ANOVA and all results were given as mean ± SEM. The level of significance was set at P < 0.05. 3. RESULTS 3.1 Direct labeling and quality control of 99mTc with UBI 29-41 UBI 29-41 was labeled with 99mTc using a simple and modified procedure according to the method of Melendez- Alafort [3]. With this labeling procedure, the complexation of 99mTc with UBI 29-41 at room temperature was quite rapid. The radiochemical purity of the labeled UBI was evaluated by Sep-Pak C-18, which successfully resolved the labeled product from reduced/hydrolyzed 99mTc and free 99mTc pertechnetate as previously reported [3]. The three labeled preparations at room-temperature and at 37 0C comprised > 80 % of 99mTc-UBI 29-41 evaluated by Sep-Pak C-18 (Table 1). Free 99mTc-pertechnetate evaluated by ITLC at room-temperature and at 37 0C revealed < 10 % (data not shown). HPLC chromatogram showed a major peak of radioactivity and ascribed to 99mTc-UBI 29-41 (Fig. 1), which showed a retention time at 15.02 ± 0.10 min (n = 6). The recovery of three formulations was > 80 % after 1-6 h of labeling (data not shown). Fig. 1 HPLC chromatogram of 99mTc-UBI 29-41 on reverse phase C-18 with on line radioactivity monitoring. 24 View slide
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 Table. 1 Radiochemical stability of 99mTc-UBI 29-41 at room temperature (n = 8-13) and at 37 0C (n = 2-3) for 0-6 h as determined by Sep-Pak C18. 99m 99m Formulation Time Tc-UBI 29-41 at Tc-UBI 29-41 at (h) room- temperature 37 0C (%) (%) 1 0 84.26 ± 3.18 - 1 83.19 ± 3.31 80.75 ± 4.42 2 84.32 ± 2.79 84.75 ± 3.65 6 83.91 ± 3.91 83.99 ± 6.70 2 0 93.44 ± 1.02 - 1 92.90 ± 1.16 89.73 ± 4.02 2 91.79 ± 0.86 90.84 ± 0.41 6 92.55 ± 0.71 92.76 ± 3.48 3 0 92.36 ± 1.63 - 1 92.85 ± 1.09 94.65 ± 1.97 2 92.03 ± 0.95 90.19 ± 2.49 6 92.68 ± 1.24 93.61 ± 0.60 3.2 Dilution stability The stability of three formulations of 99mTc-UBI 29-41 after being diluted with saline for 1-6 h showed the radiochemical purity > 70 % evaluated by Sep-Pak C-18 mini cartridge (Table 2) and free 99mTc- pertechnetate < 30 % revealed by ITLC (data not shown). Table 2. Radiochemical stability of 99mTc-UBI 29-41 diluted in normal saline at pH 7 and 9 for 1-6 h as determined by sep-pak C18. (n = 3-6). 99m Formulation Time Tc-UBI 29-41 at room temperature (h) pH 7 pH 9 1 1 81.46 ± 4.35 86.02 ± 0.98 2 82.74 ± 2.04 84.75 ± 3.39 6 86.67 ± 0.49 85.21 ± 2.63 2 1 91.94 ± 0.60 91.59 ± 0.80 2 92.21 ± 0.59 92.07 ± 0.69 6 93.75 ± 0.47 90.99 ± 2.22 3 1 95.87 ± 2.56 95.73 ± 1.22 2 97.82 ± 2.18 98.77 ± 1.23 6 94.47 ± 3.01 96.19 ± 1.66 3.3 Serum stability The radiochemical stability of 99mTc-UBI after being incubated in serum at 37 0C showed > 60 % which determined by Sep-Pak C18 (Table 3) and ITLC analysis revealed small amounts of released/free technetium-99m (< 6 % of the total activity at entire intervals) (data not shown). 25 View slide
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 Table 3. Radiochemical stability of 99mTc-UBI 29-41 diluted 1:1 in normal human serum and incubated at 37 0C for 1-6 h as determined by Sep-Pak C18. (n = 3-6). 99m Formulation Time Tc-UBI at room (h) temperature (%) 1 1 67.36 ± 6.24 2 68.55 ± 4.40 6 70.31 ± 7.89 2 1 76.06 ± 0.64 2 77.46 ± 1.88 6 80.96 ± 1.24 3 1 81.12 ± 3.71 2 79.55 ± 2.06 6 79.14 ± 3.13 3.4 Cysteine challenge The binding stability of 99mTc with UBI 29-41 was also confirmed with a cysteine challenge test at 37 0 C. The dissociation of formulation 2 and 3, after 6 h of incubation with 500 molar ratio of cysteine as free pertechnetate, were 21.94 ± 5.50 and 34.89 ±7.96 %, respectively. The percentage of dissociation was time dependence manner (Fig. 2 and 3). These results indicated the fair stability of the labeled complex in cysteine solution. The dissociation of formulation 1 in cysteine solution showed indifferently (data not shown). 1:5 5 1:50 500 Dissociation 0 30 * 25 20 15 10 % 5 0 1 2 6 Time () h Fig. 2 Dissociation of 99mTc from 99mTc-UBI 29-41 of formulation 2. Radiolabeled was incubated with cysteine to a molar ratio of 1:5 and 1:500. Each tube was incubated at 37 0C and radiochemical purity was analyzed at 1, 2 and 6 h by Sep-Pak C-18 (n=3). * The level of significance was set at P < 0.05. 26
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 1:5 5 1:500 Dissociation 50 500 40 30 20 % 10 0 1 2 6 Time () h Fig. 3 Dissociation of 99mTc from 99mTc-UBI 29-41 of formulation 3. Radiolabeled was incubated with cysteine to a molar ratio of 1:5 and 1:500. Each tube was incubated at 37 0C and radiochemical purity was analyzed at 1, 2 and 6 h by Sep-Pak C-18 (n=2). 3.5 Biodistribution of 99mTc-UBI 29-41 in normal and infected mice The tissue distribution of 99mTc-UBI in normal mice was rapidly cleared from the blood and the body of mice into the urine via the kidneys and the urinary tract as a result of its high hydrophilicity (data not shown). The accumulation of 99mTc-UBI in the thigh muscle of mice infected with S. aureus of 3 formulations showed target (T)/non-target (NT) ratio as 0.9-4.9 (Table 4, 5 and 6). The liver, spleen and lung also showed a gradual decline in uptake value with the different formulations (formulation 3 < 2 < 1). Biodistribution of 99mTc-UBI 29-41 in K. pneumoniae infected mice of 3 formulations exhibited T/NT ratio as 1.3-9.1 (Table 7, 8 and 9). The liver, spleen and lung also showed a gradual decline in uptake value with the different formulations (formulation 3 < 2 < 1). Table 4. Biodistribution of 99mTc-UBI 29-41 in S. aureus infected mice (formulation 1). Data are expressed as the percentage of total 99mTc-activity of the injected dose/gram of tissue. (n = 3). Tissue Time (h) 1 2 4 Liver 18.40 ± 0.79 12.10 ± 0.60 14.75 ± 1.77 Spleen 9.30 ± 1.04 6.49 ± 0.53 10.01 ± 2.17 Kidney 33.87 ± 4.51 20.49 ± 1.24 15.48 ± 0.31 Muscle (NT) 0.37 ± 0.035 0.25 ± 0.11 0.27 ± 0.10 Bone 1.18 ± 0.37 0.47 ± 0.03 0.53 ± 0.20 Lung 9.68 ± 1.22 4.61 ± 0.19 3.94 ± 1.71 Heart 0.51 ± 0.11 0.39 ± 0.11 0.67 ± 0.21 Blood 1.03 ± 0.079 0.43 ± 0.05 0.43 ± 0.10 Urine 463.45 ± 24.77 375.59 ± 19.98 239.57 ± 31.48 Stomach 1.48 ± 0.58 1.62 ± 0.61 2.08 ± 0.38 Intestine 1.56 ± 0.24 1.63 ± 0.11 1.93 ± 0.07 Infected muscle (T) 1.75 ± 0.38 1.03 ± 0.03 1.31 ± 0.32 T/NT 4.73 4.12 4.85 27
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 Table 5. Biodistribution of 99mTc-UBI 29-41 in S. aureus infected mice (formulation 2). Data are expressed as the percentage of total 99mTc-activity of the injected dose/gram of tissue. (n = 3). Tissue Time (h) 1 2 4 Liver 11.34 ± 2.21 7.71 ± 0.42 9.68 ± 0.76 Spleen 6.20 ± 0.90 4.00 ± 0.92 5.07 ± 0.63 Kidney 32.53 ± 4.51 22.01 ± 1.00 19.78 ± 0.58 Muscle (NT) 0.17 ± 0.06 0.41 ± 0.11 0.65 ± 0.09 Bone 0.58 ± 0.13 0.22 ± 0.08 0.24 ± 0.07 Lung 4.24 ± 0.87 2.22 ± 0.77 1.99 ± 0.27 Heart 0.20 ± 0.08 0.29 ± 0.08 0.43 ± 0.07 Blood 0.53 ± 0.10 0.21 ± 0.06 0.17 ± 0.05 Urine 257.55 ± 91.64 177.21 ± 40.30 399.68 ± 297.65 Stomach 0.84 ± 0.54 1.25 ± 0.47 0.54 ± 0.17 Intestine 1.08 ± 0.55 1.02 ± 0.14 0.97 ± 0.17 Infected muscle (T) 0.66 ± 0.34 1.33 ± 0.36 1.55 ± 0.77 T/NT 3.88 3.24 2.38 Table 6. Biodistribution of 99mTc-UBI 29-41 in S. aureus infected mice (formulation 3). Data are expressed as the percentage of total 99mTc-activity of the injected dose/gram of tissue. (n = 3). Tissue Time (h) 1 2 4 Liver 4.95 ± 0.73 4.57 ± 0.43 4.35 ± 0.29 Spleen 2.35± 0.14 3.08 ± 0.26 1.97 ± 0.25 Kidney 31.13 ± 1.90 34.31 ± 4.75 19.95 ± 0.90 Muscle (NT) 0.36 ± 0.13 0.14 ± 0.04 0.17 ± 0.05 Bone 0.21 ± 0.03 0.33 ± 0.04 0.04 ± 0.01 Lung 1.47 ± 0.14 1.43 ± 0.01 0.87 ± 0.28 Heart 0.27 ± 0.04 0.16 ± 0.05 0.34 ± 0.06 Blood 0.22 ± 0.02 0.14 ± 0.04 0.10 ± 0.04 Urine 470.33 ± 84.34 723.99 ± 236.70 400.69 ± 38.82 Stomach 0.69 ± 0.09 0.49 ± 0.06 0.40 ± 0.04 Intestine 0.89 ± 0.12 0.90 ± 0.07 0.96 ± 0.11 Infected muscle (T) 0.32 ± 0.15 0.30 ±0.15 0.33 ± 0.04 T/NT 0.88 2.14 1.94 Table 7. Biodistribution of 99mTc-UBI 29-41 in K. pneumoniae infected mice (formulation 1). Data are expressed as the percentage of total 99mTc-activity of the injected dose/gram of tissue. (n = 3). Tissue Time (h) 1 2 4 Liver 20.46 ± 0.81 20.73 ± 1.83 17.89 ± 0.69 Spleen 11.24 ± 1.02 8.70 ± 2.19 7.69 ± 0.44 Kidney 20.73 ± 1.37 17.38 ± 0.68 13.73 ± 0.75 Muscle (NT) 0.79 ± 0.56 0.34 ± 0.05 0.52 ± 0.10 Bone 0.53 ± 0.11 0.36 ± 0.12 0.67 ± 0.19 Lung 10.89 ± 3.39 7.89 ± 1.58 6.37 ± 1.03 Heart 0.66 ± 0.25 0.64 ± 0.07 0.73 ± 0.10 Blood 1.01 ± 0.09 0.78 ± 0.02 0.53 ± 0.10 Urine 743.07 ± 246.73 446.04 ± 43.63 305.87 ± 55.35 Stomach 3.25 ± 0.08 3.67 ± 0.29 2.29 ± 0.17 Intestine 1.83 ± 0.06 2.69 ± 0.35 2.77 ± 0.09 Infected muscle (T) 2.28 ± 1.36 2.17 ± 0.83 2.33 ± 0.38 T/NT 2.89 6.38 4.48 28
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 Table 8. Biodistribution of 99mTc-UBI 29-41 in K. pneumoniae infected mice (formulation 2). Data are expressed as the percentage of total 99mTc-activity of the injected dose/gram of tissue. (n = 3). Tissue Time (h) 1 2 4 Liver 8.30 ± 1.47 10.76 ± 1.46 9.84 ± 0.90 Spleen 3.42 ± 0.24 4.43 ± 1.11 3.90 ± 1.19 Kidney 25.71 ± 0.62 28.63 ± 3.40 26.30 ± 2.46 Muscle (NT) 0.46 ± 0.12 0.52 ± 0.22 0.79 ± 0.31 Bone 0.33 ± 0.07 0.34 ± 0.06 0.29 ± 0.07 Lung 4.52 ± 1.04 4.19 ± 1.92 2.22 ± 1.09 Heart 0.59 ± 0.16 0.49 ± 0.39 0.44 ± 0.16 Blood 0.34 ± 0.01 0.22 ± 0.09 0.18 ± 0.06 Urine 265.82 ± 111.45 343.12 ± 14.10 147.89 ± 7.81 Stomach 0.45 ± 0.05 0.83 ± 0.27 0.98 ± 0.17 Intestine 0.28 ± 0.11 1.10 ± 0.21 1.13 ± 0.17 Infected muscle (T) 0.58 ± 0.08 4.75 ± 1.95 1.07 ± 0.31 T/NT 1.26 9.13 1.35 Table 9. Biodistribution of 99mTc-UBI 29-41 in K. pneumoniae infected mice (formulation 3). Data are expressed as the percentage of total 99mTc-activity of the injected dose/gram of tissue. (n = 3). Tissue Time (h) 1 2 4 Liver 7.32 ± 0.34 5.07 ± 0.13 5.29 ± 0.40 Spleen 3.13 ± 0.49 2.47 ± 0.40 2.36 ± 0.39 Kidney 67.64 ± 2.04 30.90 ± 1.28 21.94 ± 1.32 Muscle (NT) 0.13 ± 0.02 0.21 ± 0.05 0.19 ± 0.01 Bone 0.45 ± 0.01 0.28 ± 0.15 0.14 ± 0.06 Lung 4.09 ± 0.50 1.72 ± 0.28 1.36 ± 0.17 Heart 0.38 ± 0.09 0.39 ± 0.02 0.29 ± 0.08 Blood 0.57 ± 0.01 0.18 ± 0.03 0.18 ± 0.02 Urine 607.90 ± 161.65 795.18 ± 316.05 1197.43 ± 660.53 Stomach 1.05 ± 0.51 1.28 ± 0.74 0.83 ± 0.10 Intestine 1.38 ± 0.35 1.76 ± 0.69 1.34 ± 0.17 Infected muscle (T) 1.06 ± 0.47 1.03 ± 0.48 1.02 ± 0.56 T/NT 8.15 4.90 5.37 4. DISCUSSION AND CONCLUSION Currently, hundreds of antimicrobial peptides have been developed. Although various antimicrobial peptides are chemically different, most of them are cationic (positively charged). Initial interaction between these peptides and micro-organisms is mediated by the cationic domains of peptides and negatively charged bacterial phospholipids and specific cell wall components, such as lipoteichoic acid. The preference of antimicrobial peptides for micro-organisms over host cells can explained by differences in charge and composition of membranes between bacterial and human cells. The characteristic properties of bacterial membranes are a large membrane potential, high level of negatively charged phospholipids and a lack of cholesterol and cationic lipids. Human cells have a low membrane potential and moderated level of cholesterol and anionic lipids [8]. The spectrum of infectious diseases has changed over the last few years, hence the requirements for radionuclide imaging for the detection of infection are becoming more demanding [9]. Inflammation can be described as the reaction of the body to any kind of injury such as trauma, ischemia and also invasion by micro- organism [10]. There were many reports showing a powerful diagnosis of scintigraphic imaging on infection and inflammation [9,10,11]. 29
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 A radiopharmaceutical used for imaging infection and inflammation accumulates in the infectious/inflammatory lesion because of the locally changed physiologic condition. An infectious/inflammatory focus is characterized by 1) enhanced blood flow, 2) enhanced vascular permeability, and 3) an influx of white blood cells [11]. The accumulation of radiopharmaceuticals in infectious/inflammatory foci depends on the local physiologic features of inflammation. Thus, scintigraphic images do not depend on morphologic changes but are based on physiochemical process in tissues. Therefore, scintigraphic techniques can also visualize infectious foci in their early phases, when morphological changes are not yet apparent. In addition, scintigraphic is non-invasive method for monitoring the efficacy of the antibiotic treatment on infections that are difficult to follow up by physical examination or invasive techniques [8,11]. Various 99mTc-labeled compounds have been developed for the scintigraphic detection of infection and sterile inflammation in humans. Currently there is considerable interest in labeling peptides with 99mTc for developing target-specific imaging agents for diagnosis of infection and inflammation [6,7]. The ideal radiopharmaceutical for imaging infection/inflammation should meet the following criteria. It should have 1) efficient accumulation and good retention in inflammatory foci, 2) rapid clearance from background, 3) no accumulation in non-inflamed tissues, 4) no side effects, 5) low cost and easy preparation and 6) the ability to discriminate infection from non-microbial inflammation [11]. Labeled peptides have become an important class of radiopharmaceuticals that can be used for diagnosis or therapy of receptor-specific pathologies. Recently, antimicrobial peptides have been proposed as new 99mTc agents to distinguish bacterial infection from sterile inflammation [7], and also, to monitor the efficacy of antibacterial agents in animals infected with bacteria [1]. UBI, a cationic peptide (Thr-Gly-Arg-Ala- Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg), has been found to be very useful for achieving stable technetium complexes in direct labeling without bifunctional coupling agent such as hynic and showed specificity with bacteria infected mice or rabbits both in vitro and in vivo [3,4,5,12]. In addition, both Arg 7 and Lys of UBI are highly specific coordination sites for the Tc(V)(O) action to produce Tc-UBI complex [3]. In this study we have successfully radiolabeled UBI with technetium according to previously reported. KBH4 and pyrophosphate used in an earlier direct labeling method were found to be unnecessary [3]. The purity of radiolabeled UBI was analyzed by HPLC, which showed radioactive peak obtained at a retention time of 15.02 ± 0.10 min corresponded with UBI (Fig. 1). Because a radiochemical yield of Tc-UBI 29-41 in these studies > 80 % was achieved (Table 1), thus we decided that no further purification or extraction steps was needed. The radiochemical stability of the labeled complex in serum was found to be 60-80 % at 1-6 h after preparation (Table 3) and free pertechnetate was less than 6 % (data not shown). This result indicated that 99m Tc-UBI 29-41 was instability in human serum at 37 0C. Interestingly, stability measurements in cysteine yielded similar results (Fig. 2 and 3). Combined with the results from serum stability and cysteine challenge test, it seems that radiolabeled 99mTc-UBI 29-41 was not stable to withstand blood components, such as cysteine or glutathione, that typically attack chelated radioisotopes. After successful labeling of UBI 29-41 with 99mTc and its characterization, we explored its biodistribution in vivo. Accumulation of 99mTc-UBI 29-41 was assessed in both normal and microorganism treated mice. Biodistribution studies indicated that 99mTc-UBI was taken up at high levels by the bladder, kidney and liver. The highest radioactivity of the complex in the kidneys was found at the initial time intervals. This is evidently due to the partial elimination of the complex in the urine shortly after administration. The high T/NT ratio in infected mice demonstrated its application in detecting infection induced by S. aureus and K. pneumoniae (Table 4-9). 5. ACKNOWLEDGMENTS We wish to express our grateful appreciation to The National Research Council of Thailand and Thai Royal Government for granting the scholarship and the International Atomic Energy Agency (IAEA) for partial financial support. We would like to thank all staff members of Isotope Production Division, Office of Atoms for Peace for providing facilities and valuable suggestion during this study. 30
  • KMITL Sci. J. Vol.7 No.1 Jan – Jun, 2007 REFERENCES [1] Nibbering, P. H., Welling, M. M., Paulusma-Annema, A., Brouwer, C. P. J. M., Lupetti, A., Pauwels, E. K. J. 2004 99mTc-labeled UBI 29-41 peptide for monitoring the efficacy of antibacterial agents in mice infected with Staphylococcus aureus. The Journal of Nuclear Medicine, 45(2), 321-326. [2] Welling, M. M., Lupetti, A., Balter, H. S., Lanzzeri, S., Souto, B., et. al. 2001 99mTc-labeled antimicrobial peptides for detection of bacterial and Candida albicans infections. The Journal of Nuclear Medicine, 42(5), 788-794. [3] Melendez-Alafort, L., de Maria Ramirez, F., Ferro-Flores, et. al. 2003 Lys and Arg in UBI: A specific site for a stable Tc-99m complex?. Nuclear Medicine and Biology, 30(6), 605-615. [4] Akhtar, M. S., Iqbal, J., Khan, M. A., Irfanullah, J., Jehangir, M., Khan, B., et. al. 2004 99mTc-labeled antimicrobial peptide ubiquicidin (29-41) accumulates less in Escherichia coli infection than in Staphylococcus aureus infection. The Journal of Nuclear Medicine, 45(5), 849-856. [5] Welling, M. M., Mongera, S., Lupetti, A., Balter, H. S., Bonetto, V. et. al. 2002 Radiochemical and biological characteristics of 99mTc-UBI 29-41 for imaging of bacterial infections. Nuclear Medicine and Biology, 29(4), 413-422. [6] Lupetti, A., Welling, M. M., Mazzi, U., Nibbering, P. H., Pauwels, E. K. J. 2002 Technetium-99m labelled fluconazole and antimicrobial peptides for imaging of Candida albicans and Aspergillus fumigatus infections. European Journal of Nuclear Medicine, 29, 674-679. [7] Welling, M. M., Paulusma-Annema, A., Baller, H. S., Pauwels, E. K. J., Nibbering, P. H. 2000 Technetium-99m labelled antimicrobial peptides discriminate between bacterial infections and sterile inflammations. European Journal of Nuclear Medicine, 27(3), 292-301. [8] Nibbering, P. H., Welling, M. M.,Van Den Broek, P. J., Van Wyngaarden, K. E., Pauwels, E. K. J., Calame, W. 1998 Radiolabelled antimicrobial peptides for imaging of infections: A review. Nuclear Medicine Communications, 19, 1117-1121. [9] Peters, A. M. 1998 The use of nuclear medicine in infections. The British Journal of Radiology,71(843), 252-261. [10] Rennen, H. J. J. M., Boerman, O. C., Oyen, W. J. G., Corstens, F. H. M. 2001 Imaging infection/inflammation in the new millennium. European Journal of Nuclear Medicine, 28(2), 241-252. [11] Boerman, O. C., Rennen, H., Oyen, W. J. G., Corstens, F. H. M. 2001 Radiopharmaceuticals to image infection and inflammation. Seminars in Nuclear Medicine, 31(4), 286-295. [12] Ferro-Flores, G., Arteaga de Murphy, C., Pedraza-Lopez, M., Melendez-Alafort, L., Zhang, Y. M., et. al. 2003 In vitro and in vivo assessment of 99mTc-UBI specificity for bacteria. Nuclear Medicine and Biology, 30(6), 597-603. 31