**3. Solubility enhancement of TokeOni and seMpai (29b) for sensitive**  *in vivo* **imaging**

As mentioned in the previous section, our research group has developed multicolor luciferin analogs for *in vivo* imaging. The luminescence activities of AkaLumine (**8e**) and seMpai (**29b**) are potentially suitable for BLI. Therefore, the usefulness of these analogs as reagents for *in vivo* BLI was evaluated in further animal experiments.

#### **3.1 Development of AkaBLI (TokeOni and AkaLuc)**

Prior to administering the reagent into the animal models, we increased the aqueous solubility of **8e** (which is inherently low) and developed an HCl salt of **8e**, AkaLumine-HCl (Tokeoni) [21]. In ultrapure water, the solubility of TokeOni was 40 mM, approximately 20 times higher than that of **8e** (2 mM). Therefore, TokeOni enable to administered to experimental animals with a smaller solution volume and higher concentration than **8e**. TokeOni/Fluc BLI was performed with significantly higher sensitivity than LH2/Fluc and CycLuc1/Fluc in the lungs [21] and brain [41] of mice. However, as the *in vitro* luminescence intensity of TokeOni/Fluc was lower than that of LH2/Fluc, it must be improved before imaging large animals such as marmosets.

Accordingly, Iwano *et al*. developed a mutant luciferase Akaluc specialized for TokeOni. They developed the artificial bioluminescence system AkaBLI, which combines TokeOni with Akaluc [42] (a mutation of 28 amino acid residues on *Ppy* luciferase). The AkaBLI luminescence intensity was approximately 10-fold higher in cells (*in vitro*), 52-fold higher in mouse lungs (*in vivo*), and 1400-fold higher in mouse brain tissue (*in vivo*) than LH2/Fluc luminescence intensity. In addition, AkaBLI detected single-cell signals from mouse lung and to quantified 1–10 cells. For large animal imaging, the authors inserted the Akaluc gene into an adeno-associated virus (AAV) vector, and introduced the recombinant AVV into the striatal neurons of marmosets. AkaBLI achieved video-rate real-time imaging of marmoset brains.

#### **3.2 BLI with seMpai**

seMpai (**29b**) was developed to improve two weak points in TokeOni: neutral pH and disturbance by hepatic background signals. As TokeOni is acidic [43], it may cause acidosis when injected; moreover, TokeOni detects the hepatic background signals, which are not detected by LH2 [43, 44]. Due to the effect of N atom, seMpai was sufficiently soluble for administration to experimental animals and 69 mM was dissolved in phosphate buffered saline (pH 7.4) [40]. In Fluc-expressing lung cancer model mice, the sensitivities of seMpai and TokeOni were not significantly different [40], but seMpai detected no hepatic background signals and seMpai BLI detected breast cancer micro-metastasis [43]. When repeated with TokeOni and LH2, this experiment was unsuccessful. Although seMpai/Fluc was less sensitive than AkaBLI for single-cell imaging, its imaging sensitivity could be improved mutant luciferases such as Akaluc.

Fukuchi *et al*. monitored the expression of brain-derived neurotrophic factor (BDNF) in Bdnf-luc transgenic mice with LH2, TokeOni, and seMpai [45]. TokeOni achieved the most sensitive BLI, and seMpai and LH2 were comparable. The result probably reflects the different abilities of the compounds to penetrate the blood– brain-barrier. This result also indicates the necessary of evaluating the imaging reagent in terms of both its luminescence activity and pharmacokinetics.

Additionally, biocompatibility such as cytotoxicity of TokeOni and seMpai has not been reported yet. No acute toxicity or adverse side effects were observed in mice when these compounds were administered at a concentration of 33 mM [40], however, preliminary experiments are recommended when using new analogs, not limited to TokeOni and seMpai.

#### **4. Conclusion**

By investigating the structure–activity relationship of luciferin analogs, researchers have developed various methods for tuning the luminescence

*Near-Infrared Luciferin Analogs for* In Vivo *Optical Imaging DOI: http://dx.doi.org/10.5772/intechopen.96760*

wavelengths of these analogs. However, the intensity of the luminescence is poorly controlled. If the luminescence intensity and structure–activity relationships could be to associated by a predictable law, we could synthesize new luciferin analogs with high luminescence intensity, and further develop an imaging technology with greater usefulness than conventional technologies.

For a practical imaging technology, both the luminescent substrate/enzyme activity and the pharmacokinetics are very important. Improving the various properties of the substrates and enzymes will enhance the sensitivity of bioluminescence imaging.
