Antibiotics and Synthetic Antimicrobial Peptide
Ciprofloxacin (CPFX; Hydrochloride, ≥98% activity) was purchased from LKT Laboratories, Inc. (MN, USA). UBI 29-41 denotes TGRAKRRMQYNRR (1693 Da). 6-Hydrazinonicotinic acid (HYNIC)-UBI 29-41 was synthesized at Toray Research Center (Tokyo, Japan, Fig. 1).
Labeling Procedure and Quality Control
99mTc-HYNIC(GH)2-UBI 29-41 was labeled as follows: 2 mg of GH kit (freeze-dried α-D-glucoheptonic acid [GH] 2.0 mg and SnCl2/2H2O 1.2 µg) was dissolved in 1 mL of 99mTcO4− (185 MBq/mL, Nihon Medi-Physics Co. Ltd.) and the sample was incubated for 10 min at room temperature to synthesize 99mTc-GH. Then, 200 µL of the 99mTc-GH solution and 200 µL of 400 µM HYNIC-UBI aqueous solution were mixed and incubated for 60 min at room temperature. 99mTc-HYNIC(Tricine)2-UBI 29-41 was labeled as follows: 125 µL of 99mTcO4− was mixed with 125 µL of 40 mg/mL tricine in 10 mM acetate buffer (pH 4). Then, 10 µL of 25 µM HYNIC-UBI aqueous solution and 6 µL of 1 mg/mL SnCl2/2H2O in 0.1 N HCl were added, and the sample was incubated for 10 min at 90°C . Following labeling, each reaction mixture was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). The sample was applied to an XBridge® C18 5 µm column (4.6 × 150 mm, Waters) attached to a chromatograph equipped with an on-line UV set at 254 nm and a NaI (Tl) crystal gamma detection system (Gabi star, Raytest, Straubenhardt, Germany). 99mTc-HYNIC(GH)2-UBI 29-41 was detected using a linear gradient of two eluents, 0.1% (v/v) trifluoroacetic acid (TFA)/water (solvent A) and 0.1% TFA/acetonitrile (solvent B), at a flow rate of 1.2 mL/min. The gradient was applied as follows: 95%–20% A in 15 min and 95% A for 5 min. 99mTc-HYNIC(Tricine)2-UBI 29-41 was detected using a linear gradient of two eluents, 0.01% (v/v) trifluoroacetic acid (TFA)/water (solvent A) and 0.01% TFA/acetonitrile (solvent B), at a flow rate of 1.0 mL/min. The gradient was applied as follows: 95%–70% A in 15 min and 95% A for 5 min.
Staphylococcus aureus 25923 (American Type Culture Collection) susceptible to CPFX (minimum inhibitory concentration [MIC] < 1 µg/mL) was used. S. aureus 25923 was cultured on Brain–Heart Infusion Agar (BHIA) for 24 h at 37°C. The suspension of colony was washed, counted by optical density, and used in the in vitro binding assay. For the in vivo assay, a stock solution of S. aureus 25923 stored at −80°C was used.
In Vitro Binding to S. aureus
Ten microliters of the preparation containing each labeled peptide and 10 µL of suspension containing 4 × 1011 cfu/mL S. aureus were added to 80 µL of a binding buffer (20 mM phosphate buffered saline [PBS] containing 0.01% Tween80 and 5 mM acetic acid, pH=5). For the serum conditions, instead of 10 µL of the binding buffer, 10 µL of mouse serum were added. The suspensions were gently mixed using a vortex mixer and incubated at 37°C for 1 h. After incubation, the tubes were centrifuged at 5000 ×g for 10 min. The supernatant was removed, and the pellet was resuspended in 100 µL of binding buffer and centrifuged again using the conditions described above. The supernatant was again removed and the radioactivity of the pellet was counted using a γ-counter. The radioactivity associated with the bacteria pellet was expressed as percentage of the total 99mTc activity added.
All of the procedures for the animal studies were approved by the Institutional Animal Care and Use Committee of Shionogi & Co., Ltd. (Osaka, Japan). Specific-pathogen-free (SPF) male ICR mice (CLEA Japan Inc., 5 weeks) were used in the infection and SPECT studies.
A 0.2-mL solution containing 30 kBq of 99mTc-HYNIC(Tricine)2-UBI 29-41 or 10 kBq of 99mTc-HYNIC(GH)2-UBI 29-41 was administered via the tail vein of mice. Animals were euthanized by exsanguination and opening of the thoracic cavity at 0.5, 2, and 3 h post injection. Organs were excised and weighed, and their activity was counted using a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/organ, Table 1).
Treatment of Animal Infections with Antibacterial Agents
Mice were anesthetized with isoflurane, and 6.0 × 106–2.0 × 107 cfu of bacteria in 0.1 mL saline were aseptically injected into the left thigh muscle of each mouse. Mice were initially subcutaneously administered CPFX 100 mg/kg q.d. or t.i.d. for 1 or 2 days. To evaluate the correlation between the accumulation of each labeled peptide and the viable bacterial count, mice received 0–100 mg/kg of CPFX for 2 days after infection q.d. (2, 24 h) or t.i.d. (2, 4, 6, 24, 26, 28 h) (Fig. 3).
At 46 h after infection, 0.2 mL of solution containing 10–30 MBq of each labeled peptide was administered via the tail vein of mice. The accumulation of each labeled peptide in the bacteria-infected site in mice was assessed by SPECT/CT (Triumph II SPECT 2H/XO SRI CT, TriFoil Imaging). Two hours after the labeled peptide injection, mice were anesthetized with isoflurane. The mice were then arranged lying face down on a SPECT/CT bed with both hind legs spread out and fixed with surgical tape. Whole body images were acquired under the following conditions; energy window: 20% at 140 keV, projection limit: 20 s, projection count: 64, rotation angle: 360 degrees. After whole body imaging, the mice were euthanized and the infected (left) and normal (right) legs were extracted. Leg-only images were then acquired using the same conditions. For the image processing, adjusted regions of interest (ROI) were drawn over the entire infected muscle (target [T]) and contralateral muscle (nontarget [NT]). The accumulation of each labeled peptide at the infection site was expressed as the ratio of the counts in the target and the nontarget muscles (T/NT).
Determining the Number of Viable Bacteria
After SPECT imaging, the entire infected thigh muscles were removed and individually homogenized in Mueller–Hinton broth. Serial dilutions of the thigh homogenate were plated on Brain–Heart Infusion Agar. The plates were then incubated for 24 h at 37°C, and the number of cfu was counted.
The differences between log cfu before and after treatment of mice with CPFX were evaluated using the Student t test. The P values were calculated, and statistical significance was accepted within 95% confidence limits. All results were reported as means and SD. The Pearson correlation coefficient (r) was used to assess the correlation between the accumulation of each labeled peptide and the viable bacterial count.