Vascular-targeted photodynamic therapy with glucose-conjugated chlorin e6 for dogs with spontaneously occurring tumours.

BACKGROUND: Photodynamic therapy (PDT) using photosensitisers is a minimally invasive treatment for malignant tumours. However, ideal photosensitisers are not yet established. Recently, we developed a new photosensitiser, glucose-conjugated chlorin e6 (G-Ce6). OBJECTIVES: To evaluate the clinical efficacy of vascular-targeted PDT (VTP), a type of PDT utilising a short drug-light interval, using G-Ce6 to treat spontaneously occurring tumours in dogs. METHODS: Five dogs with spontaneously occurring tumours (malignant melanoma: three; haemangiopericytoma: two; and squamous cell carcinoma: one) were subjected to VTP. These dogs were intravenously injected with G-Ce6 at doses of 1-3 mg/kg 5 min before laser irradiation. Tumours were superficially or interstitially irradiated using a 677-nm diode laser. RESULTS: Repeated VTP decreased tumour size, yielding complete remission in three dogs. Complications such as oedema surrounding normal tissues and fistulae were observed, and the oedema was self-limiting. The fistula was cured by debriding the necrotic tissues formed after VTP. CONCLUSIONS: Our findings demonstrate that VTP using G-Ce6 had antitumour effects in dogs with spontaneously occurring tumours.

Imaging findings and outcomes after traumatic cerebellar injury: a canine case report.

BACKGROUND: Traumatic brain injury (TBI) is a structural injury or physiological disruption of the brain induced by an external force. The cerebellum facilitates movement coordination and provides a sense of equilibrium; damage to this structure can cause a wide variety of symptoms, including ataxia or dystaxia, ocular motor dysfunction, and disequilibrium. TBIs localised to the cerebellum are rare in dogs, and the prognosis following this type of injury remains unclear. CASE PRESENTATION: A 10-year-old female Chihuahua/Dachshund-cross dog weighing 2.8 kg presented after a fall of approximately 1 m the preceding night. The dog exhibited paresis of all limbs and was recumbent with constant extensor rigidity with opisthotonos. The bilateral thoracic limb and right pelvic limb spinal reflexes were exaggerated, while the left pelvic limb spinal reflexes were normal. The menace response was decreased, and vertical nystagmus was observed. Magnetic resonance imaging (MRI) revealed a hyperintense lesion on T2weighted (W) images, fluid-attenuated inversion recovery, and diffusion-weighted imaging (DWI). Mannitol and prednisolone were administered, and the dog recovered. The bilateral pelvic limb postural reactions improved by Day 16. On Day 22, MRI revealed a decrease in the hyperintense area of the T2W images, and this lesion appeared isointense on DWI. CONCLUSIONS: In this case report, a dog with localised injury to the cerebellum that comprised a post-tentorial lesion recovered with a favourable outcome. Moreover, similar to reports in humans, DWI can help diagnose and evaluate TBI in dogs.

Lipid bubbles combined with low-intensity ultrasound enhance the intratumoral accumulation and antitumor effect of pegylated liposomal doxorubicin in vivo.

Pegylated liposomal doxorubicin (PLD) is a representative nanomedicine that has improved tumor selectivity and safety profile. However, the therapeutic superiority of PLD over conventional doxorubicin has been reported to be insignificant in clinical medicine. Combination treatment with microbubbles and ultrasound (US) is a promising strategy for enhancing the antitumor effects of chemotherapeutics by improving drug delivery. Recently, several preclinical studies have shown the drug delivery potential of lipid bubbles (LBs), newly developed monolayer microbubbles, in combination with low-intensity US (LIUS). This study aimed to elucidate whether the combined use of LBs and LIUS enhanced the intratumoral accumulation and antitumor effect of PLD in syngeneic mouse tumor models. Contrast-enhanced US imaging using LBs showed a significant decrease in contrast enhancement after LIUS, indicating that LIUS exposure induced the destruction of LBs in the tumor tissue. A quantitative evaluation revealed that the combined use of LBs and LIUS improved the intratumoral accumulation of PLD. Furthermore, tumor growth was inhibited by combined treatment with PLD, LBs, and LIUS. Therefore, the combined use of LBs and LIUS enhanced the antitumor effect of PLD by increasing its accumulation in the tumor tissue. In conclusion, the present study provides important evidence that the combination of LBs and LIUS is an effective method for enhancing the intratumoral delivery and antitumor effect of PLD in vivo.

A Pilot Study on Efficacy of Lipid Bubbles for Theranostics in Dogs with Tumors.

The combined administration of microbubbles and ultrasound (US) is a promising strategy for theranostics, i.e., a combination of therapeutics and diagnostics. Lipid bubbles (LBs), which are experimental theranostic microbubbles, have demonstrated efficacy in vitro and in vivo for both contrast imaging and drug delivery in combination with US irradiation. To evaluate the clinical efficacy of LBs in combination with US in large animals, we performed a series of experiments, including clinical studies in dogs. First, contrast-enhanced ultrasonography using LBs (LB-CEUS) was performed on the livers of six healthy Beagles. The hepatic portal vein and liver tissue were enhanced; no adverse reactions were observed. Second, LB-CEUS was applied clinically to 21 dogs with focal liver lesions. The sensitivity and specificity were 100.0% and 83.3%, respectively. These results suggested that LB-CEUS could be used safely for diagnosis, with high accuracy. Finally, LBs were administered in combination with therapeutic US to three dogs with an anatomically unresectable solid tumor in the perianal and cervical region to determine the enhancement of the chemotherapeutic effect of liposomal doxorubicin; a notable reduction in tumor volume was observed. These findings indicate that LBs have potential for both therapeutic and diagnostic applications in dogs in combination with US irradiation.

Photodynamic detection of a feline meningioma using 5-aminolaevulinic acid hydrochloride.

CASE SUMMARY: The present study describes the case of a feline meningioma that was detected using 5-aminolaevulinic acid hydrochloride (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence at surgery. An extra-axial mass in the temporoparietal region was observed by MRI. Following craniectomy and durotomy, photodynamic detection (PDD) was performed for detection of the tumour. Intratumour PpIX was detected using fluorescence spectrum evaluation and high-performance liquid chromatography. PDD revealed bright fluorescence of PpIX induced by 5-ALA, facilitating fluorescence-guided resection of the tumour tissue. Postoperative examination demonstrated an intratumour PpIX protein concentration of 16.8 nmol/g, and based on histopathological findings we diagnosed the mass as meningioma. RELEVANCE AND NOVEL INFORMATION: PDD using 5-ALA has been used to identify the surgical margins during resection of primary human brain tumours. Recently, we have reported post-mortem PDD using 5-ALA for a canine glioblastoma. To our knowledge, this technique has not been previously used for the detection and resection of feline brain tumours. Our findings suggest that PDD using 5-ALA is useful for intraoperative fluorescence-guided resection of malignant meningioma in cats.

A Basic Study of Photodynamic Therapy with Glucose-Conjugated Chlorin e6 Using Mammary Carcinoma Xenografts.

By using the Warburg effect-a phenomenon where tumors consume higher glucose levels than normal cells-on cancer cells to enhance the effect of photodynamic therapy (PDT), we developed a new photosensitizer, glucose-conjugated chlorin e6 (G-Ce6). We analyzed the efficacy of PDT with G-Ce6 against canine mammary carcinoma (CMC) in vitro and in vivo. The pharmacokinetics of G-Ce6 at 2, 5, and 20 mg/kg was examined in normal dogs, whereas its intracellular localization, concentration, and photodynamic effects were investigated in vitro using CMC cells (SNP cells). G-Ce6 (10 mg/kg) was administered in vivo at 5 min or 3 h before laser irradiation to SNP tumor-bearing murine models. The in vitro study revealed that G-Ce6 was mainly localized to the lysosomes. Cell viability decreased in a G-Ce6 concentration- and light intensity-dependent manner in the PDT group. Cell death induced by PDT with G-Ce6 was not inhibited by an apoptosis inhibitor. In the in vivo study, 5-min-interval PDT exhibited greater effects than 3-h-interval PDT. The mean maximum blood concentration and half-life of G-Ce6 (2 mg/kg) were 15.19 ± 4.44 µg/mL and 3.02 ± 0.58 h, respectively. Thus, 5-min-interval PDT with G-Ce6 was considered effective against CMC.

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.

Metformin enhances the cytotoxicity of 5-aminolevulinic acid-mediated photodynamic therapy in vitro.

The biguanide metformin is a drug widely used for the treatment of type 2 diabetes. Metformin enhances the cytotoxicity of chemotherapy by promoting the adenosine monophosphate-activated protein kinase (AMPK) autophagy signaling pathway. Photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), leads to apoptosis when PpIX accumulates in the mitochondria, and also leads to autophagy through activation of AMPK. In the present study, the effect of metformin in combination with 5-ALA-PDT was evaluated in vitro in KLN205 lung cancer cells. At a fluence of 5 J/cm2, 5-ALA-PDT in combination with 5 mM metformin exhibited significantly increased cytotoxicity compared with that observed with 0 and 0.1 mM metformin (P=0.0197 and P=0.0423, respectively). The cells treated with 5-ALA-PDT and metformin exhibited condensation of nuclear chromatin and the presence of autophagosomes. These results indicate that apoptosis and autophagy occur in KLN205 cells following combined treatment with 5-ALA-PDT and metformin. The results from the present study are the first to indicate, to the best of our knowledge, that metformin potentiates the efficacy of 5-ALA-PDT.

Bleomycin enhances the efficacy of sonodynamic therapy using aluminum phthalocyanine disulfonate.

Sonodynamic therapy (SDT), or ultrasound combined with sonosensitization, is a promising approach because it is noninvasive and penetrates deeper than light does in photodynamic therapy. We examined whether bleomycin (BLM) could improve the efficacy of SDT. We performed an in vitro study using Colon-26 cells, which are derived from mouse colon cancer. SDT with BLM was significantly more cytotoxic than SDT alone both in vitro and in vivo. We also observed an ultrasound intensity-dependent cytotoxic effect of SDT with BLM. These findings suggest that SDT with BLM might provide a novel noninvasive treatment for deep-seated tumors.

Clinical systemic lupeol administration for canine oral malignant melanoma.

Canine oral malignant melanoma (COMM) is the most aggressive malignant tumor in dogs. Lupeol is a triterpene extracted from various fruits and vegetables that reportedly inhibits melanoma cell proliferation in vitro and in vivo. In this study, the efficacy of subcutaneous lupeol for spontaneous COMM was evaluated. A total of 11 dogs (3, 5 and 3 dogs diagnosed with clinical stage I, II and III melanoma, respectively) were evaluated. Subcutaneous lupeol (10 mg/kg) was administered postoperatively at various time points to treat these 11 COMM cases. Of the 11 subjects, 7 exhibited no local recurrence 180 days postoperatively and no severe adverse effects were observed in any of the cases. Furthermore, no distant metastasis was observed during the experimental period. Therefore, systemic lupeol may prevent local tumor progression and distant metastasis and may be a novel adjuvant treatment for the treatment of COMM.

Systemic and local injections of lupeol inhibit tumor growth in a melanoma-bearing mouse model.

Melanoma is the most aggressive type of skin cancer and it is procured from activated or genetically altered epidermal melanocytes. In the present study, the tumor-suppressive effects of systemic and local injections of lupeol, a triterpene extracted from Indian lettuce (Lactuca indica), in a melanoma-bearing mouse model were evaluated. Mice were injected once with lupeol or olive oil (solvent control) subcutaneously into the skin of the back or into the tumor tissue. Seven days after the injection, the tumor growth rates were calculated and the tumor tissues were collected. Immunohistochemical staining for Ki-67 and proliferating cell nuclear antigen (PCNA) were performed. The tumor growth rates in the lupeol-injected group were significantly decreased compared to those observed in the non-treated (NT) and solvent control groups. Lupeol also significantly decreased the areas positively stained for Ki-67 and PCNA in the tumor tissues compared to those in the NT and solvent control groups. The results of the present study demonstrated that systemic and local injections of lupeol suppress tumor growth and induce cell cycle arrest in a melanoma-bearing mouse model. These data suggest that lupeol may be effective as a novel therapeutic option for melanoma patients.