Synthesis of ^99mTc-gemifloxacin freeze dried kits and their biodistribution in Salmonella typhi, Pseudomonas aeruginosa and Klebsiella pneumoniae

2.1 Materials and methods

Octanol (Sigma Aldrich, USA), D-Mannitol (Sigma Aldrich, USA), Sodium pyrophosphate (RDH, Germany), Acetone (Merck, Germany), D-Penicillamine (Sigma Aldrich, USA), Trichloro acetic acid (Fisher scientific, UK), Stannous chloride (Sigma Aldrich, USA), ⁹⁹Mo/^99mTc generator was used to get ^99mTc, PINSTECH, Islamabad, Pakistan. Gemifloxacin (Java pharmaceutical, Lahore, Pakistan), Saline (Otsuka, Pakistan), 0.22 μm filter (MS® Nylon Membrane Filters, USA) were used. Rabbits were purchased from Tollinton market, Lahore. All chemicals used were of analytical grade and used without further purification.

2.2 Gemifloxacin kit preparation

The best conditions for radiolabeling of gemifloxacin with technetium were calculated through optimization studies. It includes varying gemifloxacin concentration, stannous chloride concentration, pH, and reaction time. Tests were carried out in triplicates (n = 3). Gemifloxacin kits with maximum labeling efficiency (99 ± 0.05)% were obtained when 1 mg gemifloxacin, 300 μg D-penicillamine, 50 μg stannous chloride and 0.2 g d-mannitol were dissolved by simple stirring in 1 ml of distilled water at pH 5.5 (1 N NaOH/1 N HCl). Kits prepared were filtered by 0.22 μm filter (MS® Nylon Membrane Filters, USA) and lyophilised for 18 h. These freeze dried kits were stored at 4 °C. This whole process of kit formulation was carried out under sterile conditions at room temperature.

2.3 Radiolabeling

Sodium pertechnetate (10 mCi) was freshly eluted, drawn up by an in-house packgen ^99mTc generator, was added to freeze dried kits and incubated for 10–15 min at room temperature.

2.4 Radiochemical analysis

Radiochemical purity was tested by PC and ITLC and RP-HPLC. In paper chromatography, whatman No. 3 paper and acetone was used. It was carried out to check the percentage of unbound pertechnetate (^99mTcO₄⁻). In ITLC, saline was used as solvent. It was used to investigate the amount of hydrolyzed/reduced technetium (^99mTcO₂). In ITLC, ^99mTc-gemifloxacin travel with solvent front and hydrolyzed/reduced ^99mTcO₂ hold at origin. In HPLC analysis of ^99mTc-gemifloxacin a volume of 10 μL of the solution was injected. Nucleosil C-18 column (100 Å, 5 μm, 250 × 4 mm) was used for analytical separation as stationary phase while solvent A (0.1% TFA in H₂O), solvent B (Acetonitrile) was used as mobile phase with flow rate of 0.5 mL/min. The summary of gradient program is given in Table 1. Radio-HPLC was used for the detection of ^99mTc-complexes. NaI (Tl) scintillation detector was used for radioactivity study and it is separated by a Teflon tube from UV detector.

2.5 In vitro stability tests

In vitro stability study of ^99mTc-gemifloxacin was performed to analyse any degradation of the labeled complex at room temperature and to find out suitable time of injecting the complex in order to avoid undesired products formation (radiolysis of the labeled complex) which may accumulate in non target organs. In vitro stability of ^99mTc-gemofloxacin was performed in blood serum to detect any degradation of the complex by blood enzymes. For this, 5 ml blood was centrifuged for 10 min at 3000 rpm. Blood cells were separated from serum, ^99mTc-gemiflocacin kit was added into it, and incubated at 37 °C. Radiochemical purity of the incubated kit was analyzed from 1 h–6 h, in continuous intervals by PC and ITLC. The results are reported in Table 3 (n = 3).

2.6 Partition coefficient (Log P)

Partition coefficient was calculated in octanol/saline as organic and inorganic layers. Counts were calculated in 100 μL of both the organic and inorganic layers by well-shaped gamma ray counter. Partition coefficient values were calculated by mean (n = 3). $$\text{Log}P\text{Value}:\text{Log}(\%\text{in octanol}/\%\text{in saline})$$

2.7 Serum protein binding

Serum protein binding was calculated by adding radiolabeled kit in fresh human blood, incubating for 1 h (25 °C), incubating for 10 min at 37 °C, and centrifuged at 3000 rpm. Trichloro acetic acid (TCA10%), in equal volume as serum, added, stirred well, and centrifuged at 3000 rpm (10 min). Both supernatant and residue were counted for radioactivity (n = 3).

2.8 In vitro binding assay of ^99mTc-gemifloxacin with bacteria

In vitro binding efficiency of the complex was investigated in Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae via cylinder plate method for microbiological assay (which is used for antibiotics potency detection) (USP-27, 2004). $$\%\text{Binding to Bacteria}=\text{Counts in Pellet}/(\text{Counts in Supernatant}+\text{Counts in Pellet})\times 100$$

2.9 Biodistribution

For biodistribution, in normal rabbit, ^99mTc-gemifloxacin (1 mL) was injected through iliac vein of the rabbit prior valium anesthesia (2.0 mg/mL). Scintigraphic images were taken to locate the path of injected medicine. Biodistribution in infected rabbits was observed, by injecting 1 mL suspension (3 × 10⁸ cfu/mL) of P. aeruginosa, S. typhi and K. pneumoniae separately in thigh muscles of rabbits. When infection and the swelling started in all rabbits, the radiolabeled complex (1 mL = 2.5 mCi) was injected in the iliac veins of all the rabbits separately after valium anesthesia (1 mL). Scintigraphic images were taken at (1–4 h) and 24 h post injection by Infinia Dual Head Gamma Camera.

3.1 Reducing agent (SnCl₂) effect

It is most common reducing agent used for the reduction of pertechnetate that favors its complex formation through chelation. Amount of reducing agent has quite strong effect on the labeling yield. When its amount was increased, labeling yield was also observed to increase and subsequent decrease in the amount of free pertechnetate and colloidal form. At 50 μg, labeling efficiency was found to increase (99 ± 0.05)%. While increasing the amount of stannous chloride above 50 μg, colloidal amount started increasing and reached to a large value when amount of SnCl₂ was 300 μg with corresponding decrease in labeling efficiency.

The results are summarized in Table 2.

3.2 Gemifloxacin quantity

Gemifloxacin amount was varied between 0.5 to 3 mg to optimize for minimum quantity of gemifloxacin that can give the maximum labeling. When gemifloxacin amount was 0.5 mg labeling efficiency was 81.75% and at 1 mg labeling efficiency was reached to its maximum labeling efficiency (99 ± 0.05)%. Above 1 mg the labeling efficiency remained stable and no major impact was observed on labeling efficiency, free pertechnetate and colloidal forms (Fig. 2).

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Figure 2

Effect of gemifloxacin concentration on labeling efficiency.

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3.3 pH effect

pH has strong effect on labeling efficiency. Labeling efficiency was low at low pH. By increasing pH, labeling efficiency was also increased up till pH 5.5. After pH 5.5, labeling efficiency decreased with subsequent increase in amount of reduced/hydrolyzed (^99mTcO₂) and free ^99mTcO₄⁻ (Table 2).

3.4 Effect of incubation time

^99mTc-gemifloxacin complex was developed in a small time. After 5 min, labeling efficiency was (94 ± 0.12)%. After 15 min, highest labeling efficiency was observed and became steady after it (Fig. 3).

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Figure 3

Effect of incubation time on labeling efficiency.

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3.5 Gemifloxacin kit formulation and radiochemical Purity

After optimization studies highest labeling efficiency (99 ± 0.05)% was achieved using 1.0 mg gemifloxacin, 50 μg stannous chloride, 300 μg D-penicillamine, 0.2 g D-mannitol, and 10 mCi sodium pertechnetate at pH 5.5. When complex was characterized by ITLC, minor activity remained at origin that is reduced ^99mTc, while major part of activity moved upward that represents ^99mTc-gemifloxacin. In PC major activity remained at the origin that represents the ^99mTc-gemifloxacin and minor activity was travelled that is free ^99mTcO⁻⁴. In HPLC studies of ^99mTc-gemifloxacin a single complex was detected with retention time 11.60 min (Fig. 4) by radioactive detector which is similar to as previously reported in the literature (Shah and Khan, 2011). While the retention time of the pure gemifloxacin is 8.0 min under the same chromatographic conditions.

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Figure 4

HPLC chromatogram; showing three peaks, one main peak at 11.60 due to ^99mTc-gemifloxacin with radiochemical purity (99 ± 0.05)% and two small peaks showing free and Colloidal forms of technetium.

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3.6 In vitro stability at room temperature and in serum at 37˚C

Labeling efficiency, free ^99mTcO₄⁻ and reduced/hydrolyzed ^99mTcO₂ remained constant with some insignificant changes for 6 h both at room temperature and in freshly prepared blood serum at 37 °C (Table 3).

3.7 Partition co-efficient

Partition co-efficient study showed that the complex has hydrophilic behavior. Log P value is −3.01, suggesting low lipophilicity of the complex. $$\text{Log}P\text{value}=\text{Log}(0.09/99.91)=-3.01$$

3.8 Serum protein binding

The complex showed a moderate binding with serum protein and found 55.8% in bound form (with protein) and 44.2% in unbound form. $$\%\text{of supernatant}=\text{Counts in supern}./(\text{Counts in supern}.+\text{Counts in residue})\times 100=44.2\%$$ $$\%\text{of residue}=\text{Counts in residue}/(\text{Counts in supernatant}+\text{Counts in residue})\times 100=55.8\%$$

3.9 Biodistribution study in normal and infected rabbits

Biodistribution in normal rabbit showed that kidneys and urinary bladder is the main route of excretion of medicine. In vivo study of ^99mTc-gemifloxacin in infected rabbits was investigated in P. aeruginosa, S. typhi and K. pneumoniae. Radioactivity was counted immediately after injecting the complex in thigh muscle. At the start maximum uptake was found in the liver, kidneys and bladder but with the passage of time these organs showed less activity signifying that its wash out is time dependent. Uptake of activity in both legs muscles shows a sharp difference after 4 h. Activity uptake in P. aeruginosa was 5.84 ± 0.09%ID/g and in S. typhi was 6.06 ± 0.26%ID/g and in K. pneumoniae was 6.55 ± 1.1%ID/g at 2 h post injection; these results are better than ^99mTc-ciprofloxacin uptake value at 2 h post injection i.e. starts excreting from infection after 1 h (Tables 4, 5 and 6). Activity was not seen in stomach, suggesting stability of kits in vivo conditions i.e. no colloidal (disintegration of the kit) was formed which was the issue in labeling of quinolones in early studies (Sarda et al., 2003; Britton et al., 1997). After 24 h post injection some activity was seen at infected site indicative of good binding of ^99mTc-gemifloxacin with bacteria (Fig. 5). S. pneumonia infected rats were used in past for in vivo study of labeled gemifloxacin; we have utilized rabbits. To best of our information no previous study was reported on biodistribution of ^99mTc-gemifloxacin in Pseudomonas aeruginosa, Salmonella typhi and Klebsiella pneumonia abscess.

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Figure 5

Scintigrams showing radioactivity uptake in Salmonella typhi at 1 h (a), 2 h (b), 4 h (c) and 24 h (d) Anterior and Posterior Views.

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Scintigrams of this study are presented in Figs. 5, 6 and 7.

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Figure 6

Scintigrams showing uptake of ^99mTc-gemifloxacin in Klebsiella pneumoniae at 1 h (e) Anterior View, 2 h (f) Posterior View and 4 h (g) Anterior View.

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Figure 7

Scintigrams showing uptake of ^99mTc-gemifloxacin complex in Ps. aeruginosa infection in leg muscle of rabbit at 1 h (h) Anterior View, 4 h (i) Posterior View.

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