KR101456235B1 - Method for detecting infection of bacteria using compound comprising radioactive isotope - Google Patents

Abstract

The present invention relates to a method for detecting a bacterial infection using a nucleoside analogue comprising a radioactive isotope. According to the method for detecting a bacterial infection according to one embodiment, the nucleoside analogue labeled with a radioisotope is specifically labeled only to a bacterium, so that it is possible to monitor in real time whether or not a subject is infected with a bacterium.

Description

Method for detecting infection of bacteria using compound comprising radioactive isotope}

The present invention relates to a method for detecting bacterial infection using a compound containing a radioactive isotope.

There are limitations in that it is expensive to diagnose bacterial infection, it takes a long time to cultivate and judge bacteria, making rapid diagnosis difficult, and that a biopsy is necessary for medical and biological methods. On the other hand, diagnosis of bacterial infection through molecular imaging has the advantage of enabling quantifiable diagnosis without a biopsy within a shorter time. Herpes simplex virus type 1 thymidine kinase (HSV1-tk), which is used as a representative marker gene in molecular imaging, and a non-invasive HSV1-tk gene system using various radiolabeled compounds have been widely studied. Knowing that similar thymidine kinase is intrinsically expressed in various bacteria, fusion positron emission tomography using a ^{radiolabeled compound ([ 18} F]) in a radiopharmaceutical (FLT, fluorothymidine) was conducted to obtain bacterial infection images using it. Another radiopharmaceutical (FIAU, fluorodeoxyiodouracil) radiolabeled ^{compound ([ 125} I]) was used to clearly and quantitatively determine and diagnose bacterial infection in the body by fusion single quantum emission imaging (SPECT/CT).

One embodiment provides a method for detecting bacterial infection using a compound containing a radioactive isotope by fusion positron emission tomography (PET/CT) and fusion single quantum emission imaging (SPECT/CT).

One aspect is,

a) injecting a radioisotope-labeled nucleoside analog into a subject suspected of bacterial infection;

b) tomography of the object injected with the radioisotope-labeled nucleoside analogue;

c) It provides a method for detecting bacterial infection comprising the step of confirming whether the object is infected with bacteria.

In the present invention, a radioactive isotope-labeled compound (nucleoside analog) injected into a subject suspected of bacterial infection is metabolized by thymidine kinase contained inside the bacteria and accumulated in the bacteria. It uses the fact that it is labeled as an enemy.

As used herein, the term “thymidine kinase” refers to an enzyme that transfers the γ-phosphate group of ATP to the 5-hydroxy group of pyrimidine deoxynucleoside. In particular, bacteria are It is characterized by having thymidine kinase, which has a substrate specificity different from that of animals. Therefore, only bacteria can be specifically detected in a subject suspected of bacterial infection by using the above characteristics.

Hereinafter, the bacterial infection detection method will be described in detail for each step as follows:

The method may include injecting a radioisotope-labeled nucleoside analog into a subject suspected of bacterial infection.

The injection of the radioisotope-labeled nucleoside analog may be performed by oral administration or parenteral administration, but parenteral administration is preferable, and in case of parenteral administration, intravenous injection, subcutaneous injection, and intramuscular injection , Intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered to the subject. The administration is most preferably administered by intravenous infusion.

The appropriate amount that can be administered to the subject is an amount that does not show toxicity to the administration subject, for example, when the radioisotope-labeled nucleoside analogue is [ ¹²⁵ I] FIAU, it is preferably 1 To 30 MBq, more preferably 5 to 20 MBq, most preferably 10 to 15 MBq may be administered ^{, and in the case of [18} F]FLT, preferably 1 to 30 MBq, more preferably 3 to 15 MBq, most preferably 5 to 10 MBq can be administered.

The suspected object of bacterial infection may be any animal as long as the bacteria can be infected, and may be mammals such as primates and rodents, and birds, but is not limited thereto. According to one embodiment, the subject may be a mouse or a rat.

According to one embodiment, the bacterium that can be detected by the method is a bacterium containing a thymidine kinase gene on a genome or plasmid, and may be a bacterium expressing thymidine kinase. For example, the bacterium may be selected from the group consisting of Gram-positive bacteria such as Staphylococci species or Enterococci species, Salmonella species, Proteus species, Escherichia species, Pseudomonas species, and the like, but is not limited thereto.

The isotope-labeled nucleoside analogue may be any isotope-labeled nucleoside analogue, which may be a substrate for thymidine kinase, and may be, for example, [ ¹²⁵ I]FIAU or [ ¹⁸ F]FLT. have. Since the isotope-labeled nucleoside analog is metabolized by bacterial intrinsic thymidine kinase and accumulated in bacteria, it can be a tracer by the isotope.

Thereafter, the method may include performing tomography of the object injected with the compound and confirming whether or not the object is infected with bacteria.

According to one embodiment, the tomography may be a fusion positron emission tomography or a fusion single electron emission tomography.

In the previous step, the isotope-labeled nucleoside analogs accumulate in bacteria and become isotope-labeled. Therefore, using fusion positron emission tomography or fusion single electron emission tomography, the location of the bacteria in the bacterial infected object. And distribution can be easily detected. Confirmation of bacterial infection can be analyzed in real time by the above photographing.

According to the method for detecting bacterial infection according to an embodiment, since the compound labeled with the radioactive isotope is specifically labeled only for bacteria, it is possible to monitor whether or not the subject has bacterial infection in real time.

1 is a gel photograph showing a DNA fragment (200 bp) containing a bacterial intrinsic thymidine kinase gene obtained through PCR, where A represents a control and B represents a PCR product.
Figure 2 is a graph showing the degree of accumulation of the radiolabeled compound in bacteria, A is ^{the result of using [125} I]FIAU, B is [ ¹⁸ F]FLT as the radiolabeled compound.
3 is a photograph showing the bacterial accumulation rate of the local bacterial infection portion of a mouse injected with ^{[125 I] FIAU.}
Figure 4 is a photograph showing the bacterial accumulation rate of the local bacterial infection portion of the mouse injected with ^{[18 F]FLT.}

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the invention is not limited to these examples.

Example 1: Confirmation of the presence of intrinsic thymidine kinase in bacteria

To determine whether the bacteria ( Salmonella typhimurium) expresses intrinsic thymidine kinase GenBank No. PCR primers (Bionics Co., Ltd.) were prepared by referring to the nucleotide sequence of NP460709. Using the genome of Salmonella typhimurium as a template, the PCR primer (forward primer: 5'-aggttagatattccagtactttga-3' (SEQ ID NO: 1), reverse primer: 5'-attaaacgtagcgttcattcccgc-3' (SEQ ID NO: 2)) is used to internalize bacteria. The gene encoding thymidine kinase was amplified. PCR was performed using the GeneAmp PCR 9700 instrument of Applied Biosystems Inc., and the PCR conditions are as follows: 95°C, 15 minutes → (94°C, 30 seconds → 60°C, 1 minute → 72°C, 1 minute) Repeated 35 times → 72℃, 10min → 4℃. As shown in FIG. 1 through the PCR process, the presence of the intrinsic thymidine kinase gene was confirmed by confirming the DNA fragment containing the bacterial intrinsic thymidine kinase gene of 200 bp.

Example 2: Labeling of bacterial radioactive substances by bacterial intrinsic thymidine kinase

In Example 1, after confirming the presence of the endogenous thymidine kinase gene of the bacterium (Salmonella typhimurium ^{), [125} I]FIAU and [ ¹⁸ F]FLT, radiolabeled compounds labeled with radioisotopes as substrates for thymidine kinase, were used. Was used to confirm the activity of thymidine kinase. The bacteria were prepared by culturing one day before the test, and prepared by dispensing into 6-well culture dishes in ^{1×10 4} , 5×10 ⁴ , 1×10 ⁵ and 1×10 ^{6 colony forming units (CFU).} When dispensing, PBS was used, and [ ¹²⁵ I]FIAU was treated to be 74 kBq, and [ ¹⁸ F]FLT was 370 kBq to give an accumulation time for 10, 30, 60, and 120 minutes. After the accumulation time elapsed, the bacteria were centrifuged at 14,000 rpm for 25 minutes and washed three times with physiological saline. The bacteria were collected in a microtube and the amount of the radiolabeled compound accumulated in the bacteria was measured with a gamma counter tube. The number of individuals in each group was set to 3. As shown in FIG. 2, it was confirmed that the degree of accumulation of the radiolabeled compound in the bacteria increased linearly as the number and accumulation time of bacteria increased.

Example 3: Analysis of nuclear medicine images using radiolabeled compounds in mice infected with bacterial localization

Bacteria ( Salmonella typhimurium ) 1×10 ⁸ or 1×10 ⁹ CFU was administered to the right hip by intramuscular injection to a 6-week-old Balb/c mouse (Narabio. Korea) to construct a bacterial local infection model mouse, and 6 hours later with an inhalation anesthetic (2% isoflurane). After anesthesia, a radiolabeled compound [ ¹²⁵ I]FIAU or [ ¹⁸ F]FLT was injected by tail vein injection. The amount of the radiolabeled compound was 11.1 MBq ([ ¹²⁵ I]FIAU)/head or 7.4 MBq ([ ¹⁸ F]FLT)/head, and after 2 hours and 1 hour, single quantum emission and fusion positron emission tomography (INVEON ^TM , Siemens Preclinical Solutions, Knoxvile, TN, USA). As a result of the image analysis, as shown in FIG. 3, it was confirmed that the bacterial accumulation rate was higher in the bacterial localized infection portion of the mouse injected with ^{[125 I]FIAU compared to the non-bacterial portion.}

In addition, ^[18 F] Analysis of the existing level of ^[18 F] FLT in bacteria local infection part of the injection of FLT mouse in the area of interest (ROI), 3.187 ± 1.026 ( 1 × 10 8 CFU), 7.286 ± 2.405 ( 1×10 ⁹ CFU), and the percentage of the injected radioactivity per gram of tissue unit of 0.865±0.891 and 0.591±0.561 in the part that was not locally infected with bacteria (% ID/g) was analyzed. That is, the [ ¹⁸ F]FLT accumulation rate was ^{3.4 times that of the 1×10 8} CFU bacterial localized infection part and the 1×10 ⁹ CFU 12.3 times higher than that of the non-infected part (FIG. 4 ).

The above results indicate that [ ¹²⁵ I]FIAU or [ ¹⁸ F]FLT is effectively accumulated in bacteria, so that whether or not the mouse is infected with the bacteria can be effectively detected.

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<110> Korea Institute of Radiological & Medical Sciences <120> Method for detecting infection of bacteria using compound comprising radioactive isotope <130> PN093669 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for detecting thymidine kinase gene in Salmonella typhimurium <400> 1 aggttagata ttccagtact ttga 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for detecting thymidine kinase gene in Salmonella typhimurium <400> 2 attaaacgta gcgttcattc ccgc 24

Claims (5)

A pharmaceutical composition for the diagnosis of bacterial infection by fusion positron emission tomography comprising [ ¹⁸ F] FLT. The pharmaceutical composition according to claim 1, wherein the bacterium is a bacterium expressing thymidine kinase. The pharmaceutical composition according to claim 2, wherein the bacterium is selected from the group consisting of Staphylococci species, Enterococci species, Salmonella species, Proteus species, Escherichia species, Pseudomonas species. The pharmaceutical composition according to claim 1, wherein the subject of the bacterial infection diagnosis is a mouse or a rat. delete

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