In 2010, Tamura et al. succeeded in binding indocyanine green (ICG) to a small bubble (vesicle) comprising lipid components of the cell membrane, i.e., a liposome, and called it an ICG-modified liposome [1]. When ICG-lipo was used at the time of sentinel biopsy, it was reported to stay in the lymph node for a long period of time and was more effective for the imaging of lymph nodes than ICG alone [2]. Next, we focused on the light characteristics of ICG, i.e., light with a wavelength of 800 nm to absorb heat generation (thermal effect) [3-5] and light with a wavelength of 600-800 nm to induce active oxygen (photodynamic effect) [6], and examined the possibility of photodynamic therapy (PDT) for cancer using ICG-lipo as a photosensitizer. After confirming the safety in animal experiments and the effectiveness using tumor bearing mice, PDT using ICG-lipo was started in July 2012 for spontaneous tumor cases in dogs and cats, based on these experimental results. Simultaneously, animal experiments were continued, and it was found that this drug was effective against brain tumors when it was passed through the cerebrovascular region [7]. Furthermore, since an antitumor effect was not observed in the experiment using nude mice, it was suggested that the therapy involved immune induction. In 2014, some of these results were reported [8]. In this study, we added a drug contained in ICG-lipo and examined its therapeutic effect.

From October 2014 to the end of July 2016, 38 cases of spontaneous tumors in dogs and cats were subjected to PDT using ICG-lipo at the Tottori University Animal Medical Center and affiliated hospitals.

For the adjustment of ICG-lipo, ICG-modifiers, cholesterol, DOPC, and a methanol/chloroform mixture were added to the vial. Decompression drying was performed after dissolution, and the vial was then frozen and stored until use [1]. Next, carboplatin (carboplatin intravenous infusion 50 mg/5 ml, Nippon Kayaku Co., Ltd., Tokyo) 1 ml, bleomycin (5 mg for Bleo injection, Nippon Kayaku Co., Ltd.) 0.2 ml, vincristine (1 mg for Oncobin injection, Nippon Kayaku Co., Ltd., Tokyo) 0.2 ml, doxorubicin (10 mg for Adriacin injection, Kyowa Hakko Kirin Co., Ltd., Tokyo) 0.5 ml, Answer 20 (Zeria new drug, Tokyo) 100 times diluted solution 0.1 ml was added, and the liposome was converted using a liposome generator. The concentration of each anticancer drug was 1/10 of the amount normally administered systemically. The dosage was approximately 7 kg: 1 vial; 7-20 kg: 2 vials; 21-40 kg: 3 vials.

Treatment was undertaken by diluting the ICG-lipo solution 10 times with 10% glucose solution, administered intravenously for 40-60 minutes, and then irradiated with light using a semiconductor laser (wavelength: 810 nm, maximum output: 15 W, DL-15, Asuka Medical, Kyoto) and a rotary handpiece. The output was 2.2 W/cm² at 5 W and 4.4 W/cm² at 10 W.

With respect to the irradiation method, a vinyl pack filled with echo jelly (thickness of approximately 5 mm) was placed on the skin directly above the affected area, and the light source device was placed in contact with the vinyl pack and irradiated towards the affected area. The light source device was irradiated the vinyl pack in a circular motion. The irradiation conditions were 5 W for superficial tumors and 10 W for deep tumors (nasal, intracranial, thoracoscopic, and abdominal cavities). The irradiation interval was carried out from every day to 3 days in a week. The judgment was made every time 1 course (three weeks) was completed.

The increase or decrease in tumor volume was based on the Response Criteria in Solid Tumors (RECIST) and divided into four stages: CR, PR, SD, and PD. Judgment was made by the veterinarian in charge at the end of treatment. We also asked veterinarians who conducted the treatment to complete a questionnaire (general condition, side effects, life-prolonging effects, comprehensive evaluation) regarding this treatment.

Table 1 shows the results based on RECIST for 38 cases. The judgement for these cases was as follows: CR: 3 cases, PR: 13 cases, SD: 18 cases, PD: 4 cases. The response (CR and PR) and efficacy rates (CR, PR and SD) were 42.1% and 89.5%, respectively.

Table 1: Summary of result.

Our results confirmed that treatment using a semiconductor laser and ICG-lipo is safe and effective. As of October 2014, there were anticancer drugs, carboplatin and bleomycin. In previous cases, the response rate was 53% and the efficacy rate was 62%. The number of drugs was increased from two to four drugs in anticipation of more effects. Consequently, the response rate decreased to 42%, but the efficacy increased to 82%. PD (progression) decreased from 37% to 19%. This appears to have shown the advantage of multidrug combination by chemotherapy. In addition, until September 2014, 70% of the patients had discontinued treatment after one course, but since October 2014, only 49% discontinued treatment. However, the ratio of CR is still low at 8%, and increasing CR in the future will be a challenge.

In addition to increasing the number of anticancer drugs contained in liposomes from two to four, a solution 100 times diluted with Answer 20 (a Mycobacterium tuberculosis extract, hereinafter referred to as SSM), was added in anticipation of immune enhancement. SSM is a substance in the field of human medicine that is still being used as a "Maruyama vaccine" in paid clinical trials. The authors have also attempted to administer SSM to tumors of spontaneous onset in animals using a protocol similar to that used in human medicine. Consequently, depending on the case, the disappearance of the tumor is observed only by the regular, subcutaneous administration of SSM. There is still a lack of basic SSM data. In the future, it will be necessary to experimentally prove the effectiveness of SSM.

In the current treatment, light irradiation was carried out at 2.2 or 4.4 W / cm2. It has a power density nearly 100 times larger than the irradiation conditions under which PDT is usually administered. ICG absorbs light at 600 to 800 nm to induce active oxygen. However, it is also known that the amount of active oxygen is considerably lower than that obtained with commercially available photosensitizers (for example, rezaphylline). ICG has long been known to absorb light at 800 nm to generate heat [3-5]. It can be used as an adjuvant for hyperthermia, and the authors have conducted a basic examination and veterinary clinical application so far [9-12]. The larger the irradiation amount, the greater is the thermal effect.

 ICG was combined with liposomes in anticipation of PDT and thermal effects. Therefore, to focus on thermal effects that can have a greater effect on tumors, the irradiation power was made stronger than that generally used for PDT.

At the time of the last report, differences in the efficacy of this therapy were seen based on the type of tumor. This study yielded similar results. Lymphoma, squamous cell carcinoma, mammary tumor, lung tumor, and intranasal tumors had a response rate of more than 50%, while malignant melanoma, liver tumor, and hemangiosarcoma had a response rate of 0%. The efficacy rate was 100% for tumors other than hemangiosarcoma, and it was considered that tumor growth could be suppressed by this treatment. However, to obtain CR and PR, it is necessary to use a combination of other therapies for these tumors and to reexamine the drugs contained in them in the future.

A difference in the accumulation of ICG-lipo in tumor tissue is expected as a cause of the difference in tumor effects. One out of seven cases of lymphoma was markedly effective, and five cases were effective; the response rate was very high at 85.7%. This is considering that ICG-lipo was originally developed for the purpose of staying in lymph nodes for a long time during sentinel biopsy [2]. ICG-lipo containing anticancer drugs is highly concentrated in lymphoma tissue where lymph nodes have become tumorized and drugs contained by light irradiation are released, and it is thought that ICG-lipo exerts an antitumor effect. In addition, the small number of cases of each tumor is considered one of the causes for the difference in the effect between tumors. In the future, a study with more cases would be desirable.

Currently, chemotherapy is the only treatment for lymphoma. However, the treatment rate is approximately 70%, and some cases are ineffective. In addition, side effects of anticancer drugs may be observed. These results suggest that this treatment is very effective for lymphoma.

When the effects were compared by site of development, it was found that this treatment was more effective for body surface tumors than those of the oral and abdominal cavities. Considering the principle of this treatment, a sufficient amount of light energy is required. It is presumed that sufficient light energy was secured for the body surface tumor. In contrast, since light irradiation of the oral and abdominal cavities is performed percutaneously, there is a possibility that light has not reached the tumor tissue sufficiently. Further basic examinations on this topic will be necessary in the future.

An indication of this treatment’s efficacy is that approximately half of the veterinarians reported improvement in the general condition after treatment. Evaluation of the general condition was related to the result of the antitumor effect. That is, the case judged to be PD (deterioration) by the antitumor effect judgment was judged to be deteriorated, even in a general condition.

Side effects were observed in three cases (8%). Most of these were anaphylactic symptoms immediately after administration (respiratory urgency, itching, trembling, etc.). This is due to the activation of the complement to the liposome, since this treatment uses liposome preparation. In addition, since October 2014, the number of anticancer drugs used has been increased from two to four, but the rate of side effects has not increased. For the ICG-lipo produced, the drug concentration inside and outside ICG-lipo were the same. However, since each anticancer drug outside ICG-lipo is 1/10 of the normal amount, increasing the type of anticancer drug did not affect the living body.

A life-prolonging effect was observed in approximately 80% of cases. This was almost in agreement with the efficacy rate of the antitumor effect. The reduction or inhibition of tumor progression appears to suggest prolongation of life. In recent years, life span completion while maintaining quality of life has been given importance. This trend is gradually spreading in the field of veterinary science. Although this treatment at present is incapable of tumor removal, it has been confirmed that it suppresses tumor progression. It is presumed that if the tumor does not progress, the living body can coexist with the tumor.

Our results confirmed that treatment using a semiconductor laser and ICG-lipo is safe and effective. In particular, the response rate for lymphoma was high at 85.7%. In more than two cases of tumors, the efficacy rate was 100% except for lymphoma and hemangiosarcoma. Approximately half of the veterinarians recognized improvement in the general condition after treatment. This demonstrates the effectiveness of this therapy.

1. Suganami A, Toyota T, Okazaki S, Saito K, Miyamo K, Akutsuto Y, et al. Preparation and characterization of phospholipid-conjugated indocyanine green as a near-infrared probe. Bioorg Med Chem Lett. 2012; 22: 7481-7485.
2. Toyota T, Fujito H, Suganami A, Ouchi T, Ooishi A, Aoki A, Onoue K, et al. Near-infrared-fluorescence imaging of lymph nodes by using liposomally formulated indocyanine green derivatives. Bioorg Med Chem. 2014; 22: 721-727.
3. Chen WR, Adams RL, Heaton S, Dickey DT, Bartels KE, Nordquist RE. Chromophore enhanced laser-tumor tissue photothermal interaction using an 808-nm diode laser. Cancer Lett. 1995; 88: 15-19.
4. Chen WR, Adams RL, Bartels KE, Nordquist RE. Chromophore-enhanced in vivo tumor cell destruction using an 808-nm diode laser. Cancer Lett. 1995; 94: 125-131.
5. Chen WR, Adams RL, Higgins AK, Bartels KE, Nordquist RE. Photothermal effects on murine mammary tumors using indocyanine green and an 808-nm diode laser: an in vivo efficacy study. Cancer Lett. 1996; 98: 169-173.
6. Hirano T, Kohno E, Gohto Y, Obana A. Singlet oxygen generation due to ICG irradiation. Photomed Photobiol. 2006; 28: 15-16.
7. Suganami A, Iwadate Y, Shibata S, Yamashita M, Tanaka T, Shinozaki N, et al. Liposomally formulated phospholipid-conjugated indocyanine green for intra-operative brain tumor detection and resection. Int J Pharmaceutics. 2015; 496: 401-406.
8. Okamoto Y, Osaki T, Higashi K, Ito N, Kata T, Imagawa T, et al. Outcome from photodynamic therapy with phospholipid-conjugated indocyanine green for animal spontaneous deep-seated occurring tumors. J Jap Soc Laser Surg Med. 2014; 35: 46-50.
9. Radzi R, Osaki T, Tsuka T, Imagawa T, Minami S, Okamoto Y. Morphological study in B16F10 murine melanoma cells after photodynamic hyperthermal therapy with indocyanine green (ICG). J Vet Med Sci. 2012; 74: 465-472.
10. Radzi R, Osaki T, Tsuka T, Imagawa T, Minami S, Nakayama Y, et al. Photodynamic hyperthermal therapy with indocyanine green (ICG) induces apoptosis and cell cycle arrest in B16F10 murine melanoma cells. J Vet Med Sci. 2012; 74: 545-551.
11. Onoyama M, Azuma K, Tsuka T, Imagawa T, Osaki T, Minami S, et al. Effect of photodynamic hyperthermal therapy with indocyanine green on tumor growth using colon 26 bearing mouse model. Oncol Lett. 2014; 7: 1147-1150.
12. Onoyama M, Tsuka T, Imagawa T, Osaki T, Minami S, Azuma K, et al. Photodynamic hyperthermal chemotherapy with indocyanine green in 16 cases of malignant soft tissue sarcoma: a novel cancer therapy. J Vet Sci. 2014; 15:117-123.