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Radiopharmaceutical evaluation of Gemifloxacin-[Technetium.sup.99m] and its histopathological effects on skeletal muscle of mice.

The need for rapid diagnosis of infections position on one hand and avoid unnecessary surgery on the other hand, can be very effective in the treatment process. Currently available diagnostic methods, such as Computerized Tomography (CT-scan) and Magnetic Resonance Imaging (MRI) which had ability to provide precise images for imaging abnormalities needed to make morphological changes, the process is time consuming and cannot be useful in the early stages of infection (Kumar R et al 2008, Stokkle M et al., 2002) Expanding the use of radionuclide's, mainly is due to the ease of use, determination and high sensitivity of methods that make use of specific radionuclide (Bylund DB,Toews ML 1993). So far, a wide variety of radiopharmaceuticals have been developed for the detection of inflammation and infection but already marked leukocyte as gold standard for diagnosis of infection are known. (Mirshojaei S et al., 2011) However, because of the in vitro preparation of leukocytes is complicated and expensive and not always available to work also, direct contact with the blood products increase the risk of secondary infections in patients, have not been warmly welcomed (Beker W et al., 1996, Palestro C et al., 2001). Now a lot of progress has been made in the field of imaging anti-infective agents. In 2002, the role of nuclear medicine techniques in the diagnosis of infection was studied and then progress on the use of antimicrobial agents labeled as selective markers for the diagnosis of bacterial infections, tuberculosis and fungal increased (Wareham D et al., 2005).These radiopharmaceuticals accumulate in infected sites and are metabolized by bacteria. So far, the largest pharmaceutical group that was studied in this context is the group of fluoroquinolones (Mirshojaei S et al., 2011) the first preparation of radiopharmaceuticals from this branch is labeled ciprofloxacin with technetium. This group of drug has strong antimicrobial activity. For wide range of Gram-negative bacteria DNA gyrase is target and in many Gram-positive bacteria, topoisomerase IV is the site which drug effect on it. (Fournier B et al., 2000, Solanki K et al., 1993).In research by Syed Qaiser Shah * Muhammad Rafiullah Khan was released in December 2010 , the composition of, Gemifloxacin - technetium- 99 , introduced as a special agent for imaging of infections caused by Streptococcus pneumonia. (Syed Qaiser Shah &

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.

Radiopharmaceutical preparation

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.

RESULTS

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)

CONCLUSION

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.

ACKNOWLEDGMENTS

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.

REFERENCES

(1.) Beker w, Palestro CJ, Winship J, Feld T, Pinsky CM, & Wolf F., Rapid imaging of infection with a monoclonal antibody fragment (LeukoScan). Clin Orthop RelatRes. 1996, 329: 263-72.

(2.) Bylund DB, & Toews ML., Radiolig and binding methods: practical guide and tips. Am J Physiol. 1993; 265(5): 409-21.

(3.) Erfani M, Doroudi, A.R., & Hadisi, L., 99mTctricabonyl labeling of loxacin and its biological evaluation in S. aureus as an infection imaging agent. J. Labeled Compounds and Radiopharma, 2013; 56(12): 627-631.

(4.) Fournier B, Zhao X, Lu T, Drlica K, & Hooper DC., Selective Targeting of Topoisomerase IV and DNA Gyrase in Staphylococcus aureus: Different Patterns of Quinolone-Induced Inhibition of DNA Synthesis. Antimicrob. Agents Chemother. 2000; 44(8), 2160-2165.

(5.) Kumar R, Basu S, Torigian D, Anand V, Zhuang H, & Alavi A., Role of modern imaging techniques for diagnosis of infection in the Era of 18F-Fluorodeoxyglucose Positron Emission Tomography. Clin Microbiol Rev, 2008; 21(1): 209-224.

(6.) Mirshojaei S, Gandomkar M, Najafi R, Sadat Ebrahimi S, Babaei M, & Shafiei A, Radio labeling, quality control and biodistribuo n of 99m Tc-cefotaxime as an infection imaging agent. J Radioanalytical Nucl Chem, 2011; 287: 21-25.

(7.) Palestro C, Weiland F, Seabold J, Valdivia S, Tomas M, & Moyer B., Localizing infection whit a technetium-99m-labeled peptide: initial resuts. Nucl Med Commun. 2001; 22(6):695-701.

(8.) Qaiser Shah, S. & Rafiullah Khan, M. Radiolabeling of gemifloxacin with [technetium.sup.99m] and biological evaluation in artificially Streptococcus pneumoniae infected rats. Journal of Radioanal Nucl Chem, 2011; 288: 307-312

(9.) Solanki K, Bomanji J, Siraj Q, Small M, & Britton K., [Tc.sup.99m] "Infection ": A new class of radiopharmaceutical for imaging infection. J. Nucl. Med, 1993, 34: 119.

(10.) Stokkel M, Reigman H, & Pauwels E., Scintigraphic head-to-head comparison between 99mTc-WBCs and 99mTc-leukoScan in the evaluation of inflammatory bowel disease: A pilot study. Eur J Nucl Med Mol Imaging.2002; 29(2):251-4.

(11.) Wareham D, Michael J, & Das S., Advances in bacterial specific imaging. Braz. Arch. Bio. Tech, 2005; 48 (SPE2):145-52.

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
Article Type:Report
Date:Mar 1, 2016
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