In vitro and in vivo evaluation of 99 mTc-pyrophosphate capability to bind Staphylococcus aureus A

Introduction: Scintigrafi c imaging of infection and infl ammation is of special interest in nuclear medicine diagnostic of infectious or infl ammatory diseases. For this purpose various radiolabelled compounds have been explored. The aim: The aim of this study was to fi nd out whether 99mTc-PYP posses capability to bind to Staphylococcus aureus, and possibilities for its use in bacterial infection and infl ammation not only in non-specifi c way. Methodology: 99mTc-PYP has been used for imaging infective and non-infective skeletal diseases. Protein binding, lypophilicity measurements and in vitro binding to viable and dead bacteria of 99mTc-PYP with 3 diff erent concentrations of sodium pyrophosphate decahydrate were studied. Wistar rats were used in all biodistribution evaluations. Results: All 99mTc-PYP samples were on high radiochemical purity, with high protein binding and hydrophilic character. In vitro investigations have shown that the uptake of 99mTc-PYP to Staphylococcus aureus was depended on concentration of pyrophosphate decahydrate in the samples. Thus the highest uptake to viable Staphylococcus aureus (>30 %) was obtained in the sample with 0.10 mg pyrophosphate decahydrate/1 ml. The in vivo investigation results on rats shown increased radioactivity in the infected thigh muscle (T/NT>2.3) and intensify bone uptake (5.4 ÷ 6.9 % ID/g). Conclusion: Considering that the diagnosis of bone or joint infection remains a challenging problem, it is obvious how important is to investigate whether 99mTc-PYP could be used as a specifi c agent for bacterial infection in the axial skeleton.


INTRODUCTION
Scintigrafi c imaging of infection and infl ammation is of special interest in nuclear medicine diagnostic of infectious or infl ammatory diseases.For this purpose various radiolabelled compounds have been explored [1][2][3][4].Th e main goal of these investigations was to fi nd out some compound labelled with technetium which had also capability to distinguish infection from sterile infl ammation.Among the labelled compounds, white blood cells (leukocytes) labelled with indium-111 or technetium-99m, by use of 99m Tc -hexam-ethylpropylene amine oxime ( 99m Tc-HMPAO) could be useful in infection diagnostic in clinical practice [5][6][7][8].Recently, a new radiopharmaceutical 99m Tc-ciprofl oxacin has been developed [9][10][11][12].Compared with radiolabelled leukocyte scintigraphy, 99m Tc-ciprofl oxacin scintigraphy supposes to be clinically more effective and specifi c for bacterial infection [8], but the reliability of its use is still in the phase of investigation.
Perez et al. [13] proposed 99m Tc-Sn complex of pyrophosphate ( 99m Tc-PYP) for skeletal imaging soon aft er Subramanian et al. [14,15] demonstrated that stabile complex formed between 99m Tc and linear polyphosphates localised in skeletal lesion in higher degree than in normal bone.Some new 99m Tc-Sn complexes of polyphosphates as: ethane-1-hydroxy-1, 1-diphosphonate (EHDP), methylene diphosphonate (MDP) and 2,3-dicarboxy-propane-1, 1-diphosphonic acid (DPD) were proposed later as better agents for bone imaging, although the mechanisms of their localisation in skeletal was not completely understood [16].Meanwhile, 99m Tc-PYP was used for visualization of acute myocardial infarction, for in vivo labelling of red blood cells (with 99m Tc) for radionuclide ventriculography and blood pool scintigraphy [17].Th e aim of this study was to fi nd out whether 99m Tc-PYP posses capability to bind to Staphylococcus aureus, and possibilities for its use in bacterial infection and infl ammation not only in non-specifi c way.
Protein binding -Th e standard trichloracetic acid (TCA) precipitation method for determining the percentage of 99m Tc-PYP/1 and 99m Tc-PYP/3 bound to proteins was used [18].0.1 ml of the labelled compound was added to 1.5 ml of 12 % human albumin (12 % HA, National Blood Transfusion Institute, Belgrade) and termostated at 37°C for diff erent time intervals.Aft er incubation period, 3 ml 20 % w/v trichloracetic acid was added.Precipitate was separated from the solution by centrifugation (three times, 5 min, 3000 g) and rinsed with 0.9 % NaCl (saline).Th e radioactivity of both phases (total 99m Tc) was measured separately in γ-counter with NaI (Tl) detector.Th e radioactivity bound to HA was expressed in percent to total radioactivity of 99m Tc.
Lipophilicity measurements -All lipophilicity measurements for 99m Tc-PYP were done by solvent extraction method with noctanol equilibrated with 0.15 mol dm -3 phosphate buff ers, pH=3÷7 [19].Th e 50 μl samples of 99m Tc-PYP/1 and 99m Tc-PYP/3 (~74 kBq) were added to each of duplicate test tubes containing 1050 μl phosphate buff er.Aft er mixing 100 μl was removed from the test tubes (labelled "A 0 ").Th an exactly 1000 μl of l-octanol was added and aft er wortexing of each tube for 1 min and centrifugation at 3000 g for 5 min, 100 μl sample from aqueous phase was removed (labelled "A 1 ").Samples A 0 and A 1 were counted in gamma counter and distribution coeffi cients were calculated using equation: where cpm means counts per minute.All measurements were performed at room temperature.
In vitro bacteria binding assay -In vitro binding of 99m Tc-pyrophosphate to viable bacteria was investigated using S. aureus ATCC 25923 (approximately 2x10 8 CFU/ml, CFU-colony forming units).Under the same conditions in vitro binding to dead bacteria was study.In this purpose a vial with viable S. aureus was heated in steam (autoclaving) at 120°C under 101.325kPa (1 Atm) for thirty minutes.Aft er that the samples were plated directly on surface of suitable growth media to confi rm the dead of bacteria.Th e survival of bacteria was estimated by counting the colonies 48 hours and 4 days aft er incubation at a temperature of 37°C.As there was no grown of bacteria on incubated media, the samples were used for in vitro binding assay.
Th e samples were incubated one hour at 37°C and then centrifuged for 15 min at 3000 g.Th e supernatant was removed and the radioactivity in the tubes was determined in a gamma counter.Th e radioactivity related to pellets was expressed as: Average = (the radioactivity in pellet/Total) • 100 where Total means total radioactivity in prepared 99m Tc-PYP standards.Th e percentage binding of pyrophosphate to bacteria was the diff erence of the above: A b -A s , where A b =(Average) b was radioactivity related to bacteria and A s =(Average) s radioactivity related to saline.All measurements were performed at room temperature, three times in duplicate for each value.
In vivo bacteria binding assay -Male white Wistar rats (four weeks old, 100g) were used as a model in all animal studies.Th e suspension containing S. aureus (0.3 ml, ~2x10 8 CFU/ml) was injected into the right thigh muscle of the rats.Twenty four or forty eight hours later, 0.1 ml of 99m Tc-PYP/3 (~74 kBq) was injected via the tail vein. 1 or 4 hours aft er the injection of 99m Tc-PYP, the rats were sacrifi ced and biodistribution was determined.Samples of infected thigh muscle, contralateral normal thigh muscle, bone (femur) and blood were weighted and radioactivity was measured in gamma counter.Th e radioactivity of other organs was measured too.Th e results were expressed as the percentage uptake of injected dose per gram of tissue (% ID/g) or per organ (% ID/organ), the infected-to-normal thigh muscle ratio and the infected thigh muscle-to-blood ratio.In the same time the biodistribution of 99m Tc-PYP/1 and 99m Tc-PYP/3 was examined in control group of animals.Six animals were used for each of the experiment.All animal experiments were carried out in compliance with United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientifi c Investigations, 2 nd edn.
Statistical analysis -All data were expressed as means ± standard deviation (SD).Th e statistical analysis of biodistribution evaluation results was performed by One-way analysis of variance (ANOVA) and Independent t-test (at signifi cance levels of 0.01 and 0.05) to determine the signifi cance in the distinction between diff erent groups.

RESULTS
All three diff erent preparation of PYP (PYP/1, PYP/2 and PYP/3) labelled with 99m Tc were of high radiochemical purity (>95 %).Considering that in vitro bacteria binding experiments were performed with 99m Tc-PYP prepared with three diff erent concentrations of Na 4 P 2 O 7 •10 H 2 O and SnCl 2 •2 H 2 O, the infl uence of their concentrations on protein binding and lypophilicity results were investigated too.Th e results were presented in Table 1 and Table 2 Th e protein binding measurements have shown that the diff erences in concentration of pyrophosphate did not infl uence on protein binding measurement results.99m Tc-PYP/1 and 99m Tc-PYP/3 both exhibited high level of binding for HA, which was in accordance with literature data [20].Th e lypophilicity measurement results have shown that   The symbol represents statistical signifi cance: a-represents comparison between groups 99m Tc-PYP/3 and 99m Tc-PYP/1 at the p < 0.01 level; b-represents comparison between groups 99m Tc-PYP/3 and 99m Tc-PYP/1 at the p < 0.05 level.(ANOVA and independent ttest)

Samples
Average % binding A b -A s 2x10 8 bacteria in 1 ml saline + 0.5 ml 99m TcO 4 -A b 0.01÷1.120÷0.42 1 ml saline + 0.5 ml 99m TcO 4 -A s 0.01÷0.702x10 8 bacteria in 1 ml saline + 0.5 ml Sn (II) + 0.5 ml 99m TcO 4 -A b 89.89÷96.400÷3.64 1 ml saline + 0.5 ml Sn (II) + 0.5 ml 99m TcO 4 -A s 90.33÷92.762x10 8 bacteria in 1 ml saline + 0.5 ml 99m Tc-PYP/1 A b 1.26÷2.890÷0.88 1 ml saline + 0.5 ml 99m Tc-PYP/1 A s 1.30÷2.012x10 8 bacteria in 1 ml saline + 0.5 ml 99m Tc-PYP/2 A b 10.25÷13.76 a 8.28÷11.02 1 ml saline + 0.5 ml 99m Tc-PYP/2 A s 1.97÷2.742x10 8 bacteria in 1 ml saline + 0.5 ml 99m Tc-PYP/3 A b 37.50÷51.30b 31.15÷39.33 1 ml saline + 0.5 ml 99m Tc-PYP/3 A s 6.35÷11.97thigh muscle and blood (ANOVA and independent t-test, at signifi cance levels of 0.01 and 0.05).Th ere was statistical diff erence for spleen only at signifi cance level 0.05 for both tests, but there was no statistical diff erence for stomach (at signifi cance levels of 0.01 and 0.05).It was obvious that if the concentration of PYP was higher, accumulation in liver had of 99m Tc-PYP to killed bacteria.As bacteria uptake of 99m Tc-PYP/3 by S. aureus was the highest, in vivo behavior of only this formulation was examined in infected group of animals.In both in vitro and in vivo bacteria assays, the ratio between concentration of PYP or Sn (II) and bacteria number, were almost equal.Th e organ distribution results of 99m Tc-PYP/3 in animals infected during diff erent time and sacrifi ced 60 or 240 min aft er injection were presented in Table 6.A statistical analysis of the results for control group of animals and animals infected 24 h and sacrifi ced 60 min aft er application been the signifi cantly enhanced, but significantly reduced kidneys uptake.
In Table 4 in vitro binding to viable bacteria for 99m Tc-pyrophosphate, as well as 99m TcO 4 -and mixture of 99m Tc and Sn (II) were presented.Th ese results pointed at signifi cant infl uence of the concentration of PYP and Sn (II) on 99m Tc-PYP uptake to bacteria.While samples of 99m Tc-PYP with considerable concentration of Na 4 P 2 O 7 •10H 2 O and SnCl 2 •2H 2 O ( 99m Tc-PYP/1) have shown negligible bacteria uptake, 99m Tc-PYP/3 uptake to bacteria was high.Statistical analysis confi rmed these signifi cant diff erences in bacteria uptake (ANO-VA and t-test, at both level: p<0.01 and p<0.05).Th ere was not bacteria uptake to 99m TcO 4 -.In vitro uptake of tin (II)-99m TcO 4 -mixture was extremely high for both groups of tubes, with or without bacteria.Th e reason for this was well known hydrolysis of tin (II) in water solutions and forming of colloid [21].Th erefore, tin (II) colloid could be labelled with 99m Tc and aft er centrifugation of the mixtures, it settled at the bottom of the tubes.
In Table 5 in vitro binding results of 99m Tc-PYP/1, 99m Tc-PYP/3 and 99m TcO 4 -to dead bacteria were presented.Th ese results have shown that there was no bacteria uptake of 99m Tc-PYP/3 has shown that there was signifi cant statistical diff erence at both levels of signifi cance for spleen, kidneys, stomach, intestine, bone, infected thigh muscle and blood.Th ere was no diff erence for lungs, liver as well as non-infected thigh muscle.Th e statistical analysis of the results for control group of animals and animals infected 24 h, but sacrifi ced 240 min aft er application of 99m Tc-PYP/3 has shown that there was signifi cant statistical difference at both levels of signifi cance for lungs, liver, kidneys, bone, infected thigh muscle and Dead bacteria in 1 ml saline + 0.5 ml 99m TcO 4 -A b 0.01÷0.56-1 ml saline + 0.5 ml 99m TcO 4 -A s 0.01÷0.70 Dead bacteria in 1 ml saline + 0.5 ml 99m Tc-PYP/1 A b 1.40÷2.700.10÷0.69 1 ml saline + 0.5 ml 99m Tc-PYP/1 A s 1.30÷2.01 Dead bacteria in 1 ml saline + 0.5 ml 99m Tc-PYP/3 A b 6.54÷7.63-1 ml saline + 0.5 ml 99m Tc-PYP/3 A s 6.35÷11.97blood, but there was no statistical diff erence for spleen, stomach, intestine and non-infected thigh muscle.If two groups of animals infected 24 h, but sacrifi ced in diff erent time was compared, it could be seen that there was signifi cant statistical diff erence at both levels of signifi cance for lungs, liver, kidneys, stomach, bone, infected thigh muscle and blood, but there was no statistical diff erence for spleen, intestine and non-infected thigh muscle.Th e similar statistical analysis for animals infected 48 h was done.Th e comparisons of the uptake results for both group of the infected animals (sacrifi ced in diff erent time) and control, non-treated group, have shown that there was signifi cant statistical diff erence at both levels of signifi cance for all organs, except for noninfected tight.When two groups of infected animals, sacrifi ced at diff erent time (60 and 240 min aft er application of the radiopharmaceutical) were compared, it could be seen that there was no signifi cant statistical diff erence at both levels of signifi cance for lungs, spleen, kidneys, stomach bone and non-infected thigh muscle.Statistical diff erence for intestine, infected thigh muscle and blood was confi rmed.Th e comparisons of the thigh muscle and bone uptake for infected and non-infected animals (control group) were of special interest.Th e results point at signifi cant diff erence in the means at both levels of signifi cance.Th e higher percentage of radioactivity in the bone tissue (femur) for infected animals than in control group of animals could be the consequence of infection site on bone.
Th ere was signifi cant diff erence at signifi cance levels of 0.01 and 0.05 (ANOVA and independent t-test), between the blood uptake for infected and control group of animals.Also there was statistically signifi cantly diff erence between the blood uptakes in animals infected for diff erent time.
Th e infected-to-normal thigh muscle ratio and the infected thigh muscle-to-blood ratio were shown in Table 7.As it could be seen, the obtained results point at increased radioactivity in infected thigh muscle (T/ NT>2.3).Th ere was some diff erence in thigh muscle uptake in dependence on the time aft er infection.At signifi cance levels of 0.01 and 0.05 (ANOVA and independent t-test), the means were statistically signifi cantly different for all treated animals, but T/NT ratio was higher in 48 hours-infection animals.Th e ratio between the target and non-target thigh muscle obtained 24 h aft er infection was higher in animals sacrifi ced 4 hour aft er administration of 99m Tc-PYP than for animals sacrifi ced 1 hour aft er administration.Th e same results were obtained for 48-hours infl ection animals.At the 0.05 as well as 0.01 level, there was statistically signifi cant diff erence in thigh muscle uptake between two groups of treated animals.Th ere were no signifi cant diff erences at the level 0.05 or 0.01 between contra lateral normal tight in infected animals and tight in control group of animals.

DISCUSSION
99m Tc-PYP has been used successfully for imaging skeletal diseases, infective and noninfective, like osteomyelitis, bone tumors or metabolic bone diseases [22,23].It was used in radionuclide scanning joint imaging and evaluation of articular disease [24].Th ere were also some attempts to use 99m Tc-PYP in staphylococcal sepsis in whole pelvic osteomyelitis [25] and staphylococcus aureus meningitis associated with pyogenic infection of the sacroiliac joint [26].Th ese results have shown that radionuclid scaning with 99m Tc-PYP could be helpful.
As a bone seeking imaging agent 99m Tc-PYP is used in imaging infection and infl ammation as non-specifi c radiopharmaceuticals. Th e main uptake mechanisms of this agent could be increased vascular permeability and increased bone metabolisms [27].Besides this, the use of 99m Tc-PYP for in vitro labelling of human leukocyte is well known.In infection and infl ammation diseases the response of leukocytes occurs rapidly.Th ey migrate to sites of infection by chemotaxis and might be used to transport radiotracers to the infected area [27].As their functional behaviour did not seem aff ected by labelling, such 99m Tc-labelled leukocytes might be used for specifi c infection and infl ammation imaging [28].
In this study we didn't investigate in what extent 99m Tc-PYP bound to leukocytes.If it was assumed that some percentage of leukocytes was labelled through 99m Tc-PYP in vivo, such labelled leukocytes could enhance the uptake of radiotracer in thigh muscle and bone of infected animals.
On the other hand, in this study the capability of 99m Tc-PYP to bind viable S. aureus was estimated.It is well known that staphylococcus aureus is the leading cause of osteomyelitis and the presence of staphylococcus aureus in bone morrow cultures in a rabbit model for S. aureus osteomyelitis was found out earlier [29].Th erefore it could be presumed that the increased bone uptake in osteomyelitis was the consequence of 99m Tc-PYP uptake to S. aureus in bone morrow.

CONCLUSION
Considering that the diagnosis of bone or joint infection remains a challenging problem, it is obvious how important is to investigate whether 99m Tc-PYP could be used as a specifi c agent for bacterial infection in the axial skeleton.Th e attempts to use 99m Tc-PYP in staphylococcal infection have to be continued and comprehensive clinical studies of this well-known radiopharmaceutical in specifi c infection and infl ammation imaging ought to confi rm its diagnostic capability.
99mTc-PYP possessed hydrophilic behavior independent on formulations and pH.Th e organ distribution results of 99m Tc-PYP/1 and 99m Tc-PYP/3 in control group of animals were presented in Table3.Th e infl uence of pyrophosphate concentration on in vivo behavior of 99m Tc-PYP in control, nontreated group of rats was confi rmed.Th ere was statistically signifi cant diff erence in organ uptake for: lungs, liver, kidneys, intestine, bone,

Table 4 .
In