Efficacy of 5-Aminolevulinic Acid in Photodynamic Detection and Photodynamic Therapy in Veterinary Medicine

5-Aminolevulinic acid (5-ALA), a commonly used photosensitizer in photodynamic detection (PDD) and therapy (PDT), is converted in situ to the established photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. To extend 5-ALA-PDT application, we evaluated the PpIX fluorescence induced by exogenous 5-ALA in various veterinary tumors and treated canine and feline tumors. 5-ALA-PDD sensitivity and specificity in the whole sample group for dogs and cats combined were 89.5 and 50%, respectively. Notably, some small tumors disappeared upon 5-ALA-PDT. Although single PDT application was not curative, repeated PDT+/−chemotherapy achieved long-term tumor control. We analyzed the relationship between intracellular PpIX concentration and 5-ALA-PDT in vitro cytotoxicity using various primary tumor cells and determined the correlation between intracellular PpIX concentration and 5-ALA transporter and metabolic enzyme mRNA expression levels. 5-ALA-PDT cytotoxicity in vitro correlated with intracellular PpIX concentration in carcinomas. Ferrochelatase mRNA expression levels strongly negatively correlated with PpIX accumulation, representing the first report of a correlation between mRNA expression related to PpIX accumulation and PpIX concentration in canine tumor cells. Our findings suggested that the results of 5-ALA-PDD might be predictive for 5-ALA-PDT therapeutic effects for carcinomas, with 5-ALA-PDT plus chemotherapy potentially increasing the probability of tumor control in veterinary medicine.


Introduction
Photodynamics techniques, which utilize photosensitizers that accumulate preferentially in neoplastic and other hyperproliferative tissues, have been recently introduced to clinical practice for       Table 2 shows the summary of the subjects treated with 5-ALA-PDT. Of the 14 subjects with tumors (15 tumors), 11 tumors showed red fluorescence induced by 5-ALA. In subjects No. 5, 9, and 11, PDD was not available. Subject No. 14 did not exhibit red fluorescence. Subject No. 9 was orally administered melphalan and prednisolone; subjects No. 10 (Figures 3 and 4) and 11 ( Figures 5 and 6) were intra-arterially administered carboplatin and doxorubicin, and numbers (Nos.) 12-14 were orally administered lapatinib. Repeated PDT+/− chemotherapy effectively reduced tumor size and achieved a response rate of 60% (Table 3).        with adenocarcinoma of the left paranasal sinus on Day 1. Opacities consistent with soft tissue could be observed in the images of the left nasal cavity and frontal sinus, and the forehead bone was significantly absorbed (red circle). (b) The tumor slightly decreased in size on Day 14 (red circle). (c) Complete remission was achieved on Day 56. (d) Imaging and histological evaluation showed no progression of disease during treatment on Day 304. The dog survived for 718 days after initial presentation.

Discussion
In the present study we evaluated the use of 5-ALA-PDD in various veterinary tumors as a screen for 5-ALA-PDT and then attempted to extend the range of 5-ALA-PDT application. In the present PDD study, the sample size for each tumor type was small, and size as well as stage of each tumor were different. Therefore, we analyzed the whole sample group. The sensitivity and specificity of 5-ALA-PDT for dogs and cats combined were 89.5 and 50%, respectively. In human medicine, numerous clinical trials have indicated that 5-ALA-PDD improves the detection of bladder cancer and brain tumors. In previous reports regarding bladder cancers, the sensitivity of fluorescence cystoscopy was considered superior to that of white light cystoscopy, with the mean values of sensitivities and specificities for fluorescence cystoscopy being 94.4% (range: 87-97) and 51.3 (range: 35-66.6), respectively [22][23][24][25][26][27][28]. In previous reports regarding brain tumors, 5-ALA-induced PpIX fluorescence improved the results in high grade glioma surgery for gross total resection. PpIX fluorescence induced by 5-ALA was observed in sarcomas as well as carcinomas, with the mean values of sensitivities and specificities for fluorescence cystoscopy being 86.8% (range: 75-92) and 85.2 (range: 71-92), respectively [29][30][31][32][33].
The results of PDD in veterinary medicine thus appear similar to those obtained in human medicine. In our study, multiple false positive samples were identified. Accordingly, it was considered that PpIX fluorescence could also be detected in benign tumors and non-neoplastic lesions. In addition, it had previously been reported that false positive fluorescence was related to inflammatory cell infiltration [13], which was consistent with the consideration that some masses with inflammation might have been present in the current study. We also identified some false negative samples. False negative fluorescence might be attributed to structure barriers, such as blood or necrotic tissues [34]. In particular, we did not detect 5-ALA-induced fluorescence in hemangiosarcoma. Previously, it has been suggested that 5-ALA induced fluorescence might be limited in cases of low tumor cell density [35]. In the present study, we did not have a direct comparison for the fluorescence intensity of PpIX of each tumor tissue. However, we previously reported that cell density strongly correlated with PpIX photon counts of mammary gland tumor tissue in dogs [17].
To date, 5-ALA-PDT has been indicated for the treatment of feline superficial squamous cell carcinoma and canine lower urinary tumor [18][19][20]. Based on the results of PDD in the present study, PpIX fluorescence was observed in 88.8% of additional malignant tumors types analyzed. Therefore, we examined the efficacy of 5-ALA-PDT for various tumors in dogs and cats. Tumors exhibiting PpIX fluorescence induced by 5-ALA were responsive to 5-ALA-PDT. Although a single PDT application was not curative, repeated PDT+/− chemotherapy achieved long-term tumor control in these subjects. Conversely, 5-ALA-PDT did not have a therapeutic effect for subject No. 14, in which PpIX fluorescence was not observed. Specifically, PDT combined with intra-arterial chemotherapy was safe, well tolerated, and effective for the treatment of paranasal sinus and lower urinary tract tumors in dogs and may therefore represent a suitable alternative to conventional therapy.
Finally, to elucidate the mechanism underlying the cytotoxicity of 5-ALA-PDT in vitro, we compared the cytotoxicity and intracellular PpIX concentration using various primary tumor cells. Our in vitro study showed that the cytotoxicity induced by 5-ALA-PDT correlated with intracellular PpIX concentration in carcinomas, which is consistent with a previous report that the cytotoxicity for ovarian clear-cell carcinoma was correlated with the intracellular PpIX [35]. Based on the current findings, it was considered that the therapeutic effects of 5-ALA-PDT for carcinomas were potentially correlated with fluorescence brightness of PpIX induced by 5-ALA in clinical cases. However, we found that LuBi cells, in which PpIX was accumulated at moderate level, were not killed by 5-ALA-PDT. As glutathione peroxidase activity has been reported to contribute to the resistance of tumor cells to PDT [36], it was considered that the 5-ALA-PDT resistance of LuBi cells might be related to such activity. However, the cytotoxicity induced by 5-ALA-PDT did not correlate with intracellular PpIX concentration in sarcomas. Therefore, the cell death mechanism by 5-ALA-PDT in sarcomas remains to be elucidated. In future studies, we will analyze the detailed mechanism underlying the 5-ALA-PDT resistance of these cells.
In carcinomas, the intracellular PpIX concentration showed a strong negative correlation with the expression of FECH mRNA levels but did not show a correlation with the expression of UROS mRNA. It has been reported that the expression of FECH protein played an important role in PpIX accumulation in human bladder cancer cells [37]. In addition, the levels of FECH mRNA expression in glioblastoma were also found to be lower than those in normal brains [38]. Therefore, the suppression of FECH might contribute to the intracellular accumulation of PpIX in carcinomas. In comparison, in sarcomas, the intracellular PpIX concentration showed a strong positive correlation with the relative UROS mRNA levels but did not show a correlation with the expression of FECH mRNA. To our knowledge, there have been no reports on the correlation between the relative UROS mRNA levels and the intracellular PpIX concentration in human and murine cells. It was considered that the difference in sensitivity to 5-ALA-PDT between carcinomas and sarcomas might be correlated with the differences of the mRNA expression levels of transporters and heme synthesis enzymes of 5-ALA. In future, there is need to be further examined the correlation between the intracellular PpIX concentration and these mRNA levels.

Animals
For the PDD study, we enrolled 124 client-owned dogs and cats with masses, which were brought to the Tottori University Veterinary Medical Centre between December 2011 and November 2018. The masses were resected under general anesthesia to provide a definitive diagnosis. A total of 109 dogs (male: 31, castrated male: 14; female: 35, spayed female: 29) were utilized in the age range of 0.9-16 years (mean and median, 10.3 and 11 years, respectively) and weighing between 2.1 and 53 kg (mean and median, 13.2 and 9.6 kg, respectively). In addition, 15 cats (male: 2, castrated male: 3; female: 5, spayed female: 5) were used in the age range of 7-15 years (mean and median, 10.8 and 10 years, respectively) and weighing between 2.7 and 6.6 kg (mean and median, 4.3 and 4.5 kg, respectively).

PDD Using 5-ALA
The dogs and cats were orally administered 5-ALA HCl at a dose of 40 mg/kg 4 h prior to biopsy or surgery. Generally, butorphanole (0.2 mg/kg) and midazolam (0.15 mg/kg) were administered as pre-anesthetic agents and general anesthesia was induced with intravenous propofol (4 mg/kg), and then maintained with isoflurane and oxygen after tracheal intubation. Furthermore, robenacoxib (Onsior ® , Elanco Japan, Tokyo, Japan) was administered subcutaneously at a dose of 2 mg/kg as an analgesic agent. After excision of 141 masses with surrounding normal tissues, the masses were further incised through the midline and the non-necrotic areas were fluoresced using an LED light source at 405 nm. The video camera (HDR-CX180, SONY, Tokyo, Japan) was equipped with a long-pass filter designed to block blue light (for observation of fluorescence). For fluorescence spectrometry, LED405-SMA-TI (Thorlabs Japan Inc., Tokyo, Japan), R600-8-UV-VIS-SR (StellarNet, Tampa, FL, USA), and Black-Comet CXR-50 TEC spectrometers (StellarNet) were used to obtain the spectra for each tissue. The maximum fluorescence peak of PpIX was at approximately 635 nm. The PDD protocol was approved by the Ethics Committee at the Faculty of Agriculture, Tottori University (ethical approval number: H28-003).

Histology
To analyze sensitivity and specificity, samples from fluorescent and non-fluorescent tissues were evaluated histologically. The mass tissues were fixed in 4% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Animals
For the PDT study, we enrolled 14 client-owned dogs and cats with tumors, which were brought to the Tottori University Veterinary Medical Centre between November 2011 and May 2018. Specifically, 10 dogs (male: 4, castrated male: 1; female: 3, spayed female: 2) were utilized in the age range of 6-15 years (mean and median, 10.3 and 10.5 years, respectively) and weighing between 2.8 and 30 kg (mean and median, 13.9 and 11.5 kg, respectively). In addition, 4 cats (female: 3, spayed female: 1) were used in the age range of 8-13 years (mean and median, 10.7 and 11 years, respectively) and weighing between 3.3 and 4.6 kg (mean and median, 4.1 and 4.4 kg, respectively). Each tumor was diagnosed by histopathological examination of biopsy samples.

PDT Using 5-ALA
A total of 10 dogs and 4 cats with tumors (15 tumors) were orally administered 5-ALA at a dose of 40 mg/kg 4 h prior to irradiation. Simultaneously, the dogs were subcutaneously administered 1 mg/kg maropitant (Zoetis Japan, Tokyo, Japan). The patients were treated under sedation or general anesthesia. For sedation, medetomidine (0.03 mg/kg) and midazolam (0.15 mg/kg) was intramuscularly administered. For general anesthesia, butorphanole (0.2 mg/kg) and midazolam (0.15 mg/kg) were administered as pre-anesthetic agents and general anesthesia was induced with intravenous propofol (4 mg/kg), and then maintained with isoflurane and oxygen after tracheal intubation.
The tumors were irradiated with a Ceralas 630-nm PDT diode laser (Biolitec, Jene, Germany) or 635-nm LED. For superficial lesions, LED and the quartz fiber fitted with a microlens were set toward the surface of the tumor. For interstitial irradiation, an over-the-needle intravenous catheter (14-gauge needle) was inserted at the desired location in the tumor mass. A 1-cm cylindrical diffuser fiber was inserted through an external cylinder of the catheter that had been left in place to provide support to the fiber. The response to PDT was evaluated 1 month after the first PDT according to RECIST criteria. Subsequently, the patients that were responsive to PDT were repeatedly treated by PDT once every week. The PDT protocol was approved by the Ethics Committee at the Faculty of Agriculture, Tottori University (ethical approval number: H28-004). Eight animals (Nos. 1-8) with tumors were treated with PDT only. Six dogs (Nos. [9][10][11][12][13][14] with tumors were treated with PDT and chemotherapy.

Cell Culture Conditions
A total of 15 canine tumor primary cell lines which were established by author were used in this study (Table 5) [39].