Radiopharmaceutical evaluation of Gemifloxacin-[Technetium.sup.99m] and its histopathological effects on skeletal muscle of mice.
Muhammad Rafiullah Khan 2011)
In this study, the effect of Gemifloxacin on Escherichia coli, as one of the most common infectious agent, and its histopathological effects on skeletal muscle have been examined. To investigate damage to muscle tissue, Desmin as one of the effective factors in the contraction can be studied. This protein is one of the intermediate filaments; create scaffold around the lines (Z) and attach them into the cell skeleton under the plasma membrane as well as keeps muscle fiber's sides together. So that is being an effective on contracting process. Desmin also have an impact on the proper functioning of mitochondria and is not detectable by conventional staining methods. To investigate it, the immunohistochemical techniques are used. (Desmin 2013)
MATERIALS AND METHODS
All materials used, were manufactured by Merck and Sigma. For immunohistochemical staining Nova link products were used.
All institutional and national guidelines for the care and use of laboratory animals were followed.
For the quality control radiopharmaceutical, model JASCO 880-PU HPLC apparatus with multi wave length detector and gamma- ray test-gabi detector with column cc2504.6 nucleosil 120/5 c18 with the following gradient system was used.
Mobile phase A: solution containing 0.1% TFA (tri- chloro acetic acid) in distilled water. Mobile phase B: acetonitrile 100 % Gradient system as follows:
Zero minute A 95%, B5% - five minutes A95%, B5% - five minutes A0%, B100% -30 minutes A0%, B100%.
Gamma counter EG & G / ORTEC / Model 4001M was used to determine activity.
For taking pictures Gamma camera, models Siemens smallarea mobile device was used.
[Technetium.sup.99m]from internal home generator [Molybdenum.sup.99]/[Te.sup.99m], was achieved.
10 ml of distilled water was mixed with 100 ml of hydrochloric acid so 0.1 normal hydrochloric acid was obtained. We solved 0.0031 grams of Sncl2 in 10 ml of hydrochloric acid 0.1 normal. 320 milligram (mg), Gemifloxacin in 10 ml of deionized water twice distilled solved, Then 62.5 micro liters ([micro]l) of the above solution, which is equivalent to 2 mg of the drug, with 32.25 [micro]l of Sncl2 solution equivalent to 100 mg Sncl2, mixed and 1 ml of radioisotope added to it, until 15 minutes, it was shaken every 2 minutes.
Evaluation of labeling Performance
Solution obtained with the features mentioned was analyzed by HPLC.
Evaluation of stability
100 [micro]l of labeled antibiotic added to 1 ml of human serum and leave it for 60 minutes in a water bath at 37 [degrees] Centigrade (C), Then 200 [micro]l of serum added to 200 [micro]l of alcohol, mixed and centrifuged at 15,000 revolutions per minute (rpm) for 5 minutes. After sinking a denatured protein, the supernatant analyzed by HPLC with features mentioned.
Determining the amount of labeled antibiotic binding to bacteria
100 [micro]l labeled bacteria was added to 1 ml of antibiotic at a concentration of 1 x [10.sup.8] colony forming unit (CFU), and for 60 minutes, put it in a water bath at 37 [degrees] C then 200 [micro]l alcohol solution added into a 200 [micro]l mixture of bacteria then centrifuged at 1500 rpm for 5 minutes. After sinking the supernatant denatured proteins took the activity from supernatant and lower fluid by following gamma counter: EG & G / ORTEC / Model 4001M.
Biodistribution and imaging studies in animal body
100 [micro]l of a suspension containing 1 cfu bacteria were injected into the leg muscle of mice. 24 hours after injection of bacteria, 100 ml of a solution containing 0.5 millicurie (mCi) of radio labeled antibiotics were injected via the tail vein. 1, 4 and 24 hours after an antibiotic injection , binary groups of mice were anesthetized under CO gas and follow Single Photon Emission Compute Tomography (ESPECT) Imaging was performed. Mice dissected and organs of interest, such as lung, stomach, spleen, intestine, liver, kidney, infectious and non- infectious muscle and ... Was collected and the average percentage of injected dose per gram (%ID/g) of each organ was calculated.
Preparation of histological sections and histopathological evaluation
After remove the capsule and fascia, infectious muscle and non- infectious, fixed in 10% neutral formalin solution, dehydrated in graded alcohol and embedded in paraffin. Fine sections obtained were stained with (H&E) and mounted on glass slides for light microscopic analyses.
Desmin intermediate filament by Nova link polymer detection system was used, according to kit instructions.
At the end, slides were examined by light microscopy.
Gemifloxacin structure with [C.sub.18][H.sub.20]F[N.sub.5][O.sub.4] formula and molecular weight of 389.381 g/ mol, have shown in Fig. 1.
Labeling efficiency of Gemifloxacin by Technetium by using of 100 mg Sncl2, 2 mg antibiotic, 23.7 mCi Pertechnetate (Tc[o.sub.4.sup.-]) and PH between 4-5, 96.94 % was obtained. (Fig. 2) the first peak, Pertechnetate, released in time of 5:46 minutes and the second peak is related to labeled Gemifloxacin, released in 14.55 minutes.
One hour after labeling, stability of radiopharmaceuticals in serum was 32.96%.
Labeled antibiotic binding to bacteria an hour after labeling was equal to 95.43%
Biodistribution of labeled Gemifloxacin in animal models:
Considering the numbers on the graph in Figure 3, it is clear that the percentage of injected dose per gram (%ID/g) of infectious muscles in 1 , 4 and 24 hours after injection in compare with healthy muscles respectively are , 1.49 , 1.89 and 2.36 times more, which shows the greater tendency of the drug to the infected tissue. Its high levels in the liver and kidney also suggests excretion pathway of the drug.
Imaging results from animal models
Pictures 4, 5 and 6 have been taken respectively, 1, 4 and 24 hours after injection. Warmer colors (blue to yellow) indicate the presence and extent of radiation. Sites of infection are shown with green circle, as is known, the best time for imaging is between 1 to 4 hours after injection of the radiopharmaceuticals.
24 hours after injection, due to drug elimination and 6 hours half-life of radioisotopes, no range of warm colors in the picture presents. Figure 3 The graphs of pharmaceuticals biodistribution
Results from studies of tissue sections
Desmin intermediate filaments in all samples were found positive and brown.
Changes will be classified in accordance with Table 1.
As is evident in Table 2 is not to harm healthy tissue and the extent of tissue damage in infected muscle is less over time, after effect and dispose of drug.
Obtained images by light microscope
Figure 7: healthy muscles 1 hour after injection, Healthy Myocytes (arrowhead) with adjacent nucleus. Cross section (H & E 640X)
Figure 8: infectious muscle 1 hour after injection, necrosis of muscle cells (arrowhead) and multi-core inflammatory cell infiltration (arrow) are seen with bacterial colonies (H & E 640X)
Figure 9: infectious muscle 4 hours after injection, necrosis of muscle cells (arrowhead) and multi-core inflammatory cell infiltration (arrow) are seen with bacterial colonies (H & E 640X)
Figure 10: infectious muscle 24 hours after injection, necrosis of muscle cells (arrowhead) and multi-core inflammatory cell infiltration (arrow) are seen with bacterial colonies. Injuries sustained significantly decreased (H & E 640X)
Fig. 11: healthy muscle, 4 hour after injection, Desmin intermediate filament (arrowhead) is seen in Myocytes (IHC 640X)
Figure 12: infectious muscle 1 hour after injection, Desmin intermediate filament (arrowhead) within the Myocytes that surrounding by inflammatory cells (arrow) is seen. (IHC 640X)
Figure 13: infectious muscle, 4 hour after injection, Desmin intermediate filament (arrowhead) within the Myocytes that surrounding by inflammatory cells (arrow) is seen. (IHC 640X)
Figure 14: infectious muscle 24 hours after injection, Desmin intermediate filament (arrowhead) within the Myocytes that surrounding by inflammatory cells (arrow) is seen. (IHC 640X)
At the first hour after injection highest uptake, respectively, in the liver, kidney, blood, spleen, lung and heart by amount of, 18.48, 14.26, 8.42, 5.63, 4.96 and 3.41 (%ID/g) were observed. Also uptakes in normal and infected muscle, respectively, were 1.1 and 1.64 (%ID/g). This biodistributions has many similarities with the survey by Syed Qaiser Shah and Muhammad Rafiullah Khan (2011) that following results were reported:
One hour after injection highest uptake, respectively, in the kidney, liver, blood, infected muscle by amount of, 18.50, 12.00, 11.35, 9.75, 7.85(%ID/g) were observed. They have introduced the combination of Gemifloxacin - 99mTc as an appropriate imaging agent for infections caused by Streptococcus pneumonia. Type text or a website address or translate a document. Cancel
In research by Erfani Mostafa et al., (2013), on combination of ofloxacin - 99mTc, highest uptake, respectively, in the liver, kidney, lung, spleen, and heart by amount of, 17.57, 16.47, 7.79, 4.43, 3.75, 3.55 and 0.84 and 1.96 (%ID/g) in normal and infected muscle were reported. he also suggest the best time for take a picture between 1 to 4 h after injection and he has proposed that combination is an appropriate Radiopharmaceutical to imaging of infections which have introduced by Staphylococcus aureus. These findings are also in good agreement with what we've achieved.
New antibiotics were evaluated in this study, the findings showed broad-spectrum antibiotics with high efficiency has the potential to be labeled by radioisotope [Tc.sup.99m]. Also after labeling and intravenous injection, the internal infection points of gram-negative bacteria, Escherichia coli, which cause many infections, can be diagnosed. Results of histological studies also approved infection points and were accordance with imaging results. Over time and after injection of the radiopharmaceutical, histological changes reduced so these finds can be performer in future studies on effectiveness of antibiotics and its role.
The use of new antibiotics and its labeling has good performance in the detection of infectious points and histological studies verify production process and its effectiveness.
I would like to express my appreciation to Dr Mostafa Erfani, Dr Pejman Mortazavi, Dr Iraj Pousty, Mr Mohammad Mazidi and Nuclear Science Research School, Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of Iran (AEOI), Tehran, Iran, because of their Sincere help and guidance.
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Mahmood Rekabgardan (1) *, Mostafa Erfani (2) and Mortazavi Pejman (2)
(1) M.Sc in Veterinary Histology, Faculty of Veterinary, Department of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
(2) Department of Basic Science, Faculty of Veterinary, Science and Research Branch, Islamic Azad University, Tehran, Iran.
(Received: 03 July 2015; accepted: 10 September 2015)
* To whom all correspondence should be addressed.
Caption: Fig. 1. Gemifloxacin Structure
Caption: Fig. 2. Labeling efficiency of Gemifloxacin by technetium
Caption: Fig. 3. The graphs of pharmaceuticals biodistribution
Caption: Fig. 4. 1 hour after injection
Caption: Fig. 5. 4 hour after injection
Caption: Fig. 6. 24 hour after injection
Caption: Fig. 7. Healthy muscle 1 hour after injection
Caption: Fig. 8. Infectious muscle 1 hour after injection
Caption: Fig. 9 Infectious muscle 4 hours after injection
Caption: Fig. 10. Infectious muscle 24 hours after injection
Caption: Fig. 11. Healthy muscle, 4 hour after injection
Caption: Fig. 12. Infectious muscle 1 hour after injection
Caption: Fig. 13. Infectious muscle, 4 hour after injection
Caption: Fig. 14. Infectious muscle 24 hours after injection
Table 1. Histopathological scoring in tissue samples Classification Description Value of changes Necrosis and No necrosis / inflammation 0 inflammation Local necrosis / inflammation, 1 less than 25 % Local necrosis/inflammation, between 2 25 % -50 % Necrosis/inflammation extended but 3 local Complete tissue necrosis / inflammation, 4 wide and across The results of the sections review is given in Table 2 Table 2. The results of sections review Observations Extent of Extent of necrosis Inflammation Normal Mice No1-1h after 0 0 tissue injection, normal muscle Mice No1-4h after 0 0 injection, normal muscle Mice No1-24h after 0 0 injection, normal muscle Mice No2-1h after 0 0 injection, normal muscle Mice No2-4h after 0 0 injection, normal muscle Mice No2-24h after 0 0 injection, normal muscle Infected Mice No1-1h after injection 2 4 tissue infected muscle Mice No1-4h after 1 3 injection, infected muscle Mice No1-24h after 1 2 injection, infected muscle Sum Desmin Normal Mice No1-1h after 0 + tissue injection, normal muscle Mice No1-4h after 0 + injection, normal muscle Mice No1-24h after 0 + injection, normal muscle Mice No2-1h after 0 + injection, normal muscle Mice No2-4h after 0 + injection, normal muscle Mice No2-24h after 0 + injection, normal muscle Infected Mice No1-1h after injection 6 + tissue infected muscle Mice No1-4h after 4 + injection, infected muscle Mice No1-24h after 3 + injection, infected muscle
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|Author:||Rekabgardan, Mahmood; Erfani, Mostafa; Pejman, Mortazavi|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Mar 1, 2016|
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