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Aim: We undertook studies to determine the lethal dose 50 (LD50) of acridine orange (AO) using mice in order to confirm the safety of intravenous administration of AO., Materials and Methods: We used 40 mice and AO was administered once intravenously. General behavior and mortality were continuously observed for 14 days. At the end of the experiment, all animals were sacrificed for subsequent studies., Results: The LD50 for AO in male and female mice was determined to be 32 mg/kg and 36 mg/kg, respectively. Histopathological abnormalities were observed in only one mouse which died three days after the administration of AO. The other nine mice which died immediately after the administration of AO had no pathological findings in major organs., Conclusion: The clinical use of AO can be kept at 1.0 mg/kg or below and, therefore, intravenous administration of AO might be safe for use as cancer therapy., (Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

During the past 20 years, we have found that acridine orange (AO) selectively accumulates in musculoskeletal sarcomas in vivo or exerts selective cytocidal effects against sarcoma cells in vitro after illumination of the tumor cells with visible light or irradiation of the cells with low-dose X-rays. Based on the data obtained from basic research, we have employed reduction surgery followed by photo- or radiodynamic therapy using AO (AO-PDT & RDT) in 71 patients with musculoskeletal sarcomas, in an attempt to maintain excellent limb function in the patients. We have obtained good local control rates and remarkably better limb functions with this approach as compared to the results obtained with the conventional wide resection surgery. Our basic research demonstrated that AO accumulates densely in intracellular acidic vesicles, especially lysosomes, in an acidity-dependent manner. In cancer cells that proliferate under hypoxic conditions or with Warburg's effect, active glycolysis produces an enormous number of protons, which are released by the cells via proton pumps into the extracellular fluid or lysosomes to maintain a neutral pH of the cytosolic fluid. Cancer cells contain many strongly acidic lysosomes of large sizes; therefore, AO shows marked and prolonged accumulation in the acidic lysosomes of cancer cells. Photon energy excites the AO resulting in the production of activated oxygen species, which oxidize the fatty acids of the lysosomal membrane, resulting in the leakage of lysosomal enzymes and protons, followed by apoptosis of the cancer cells. Based on these observations, we conclude that AO-PDT & RDT target acidic vesicles, especially the lysosomes, in cancer cells, to exhibit selective anti-cancer cell activity. Therefore, it is suggested that AO excited by photon energy has excellent potential as an anticancer "Magic Bullet".

Background and Objectives: We recently developed and established a new surgical therapy combining photodynamic surgery and radiodynamic therapy using acridine orange (AO) therapy after marginal or intralesional tumor resection, providing excellent limb function to sarcoma patients. The present study evaluated local recurrence rate and limb function using Disability of Arm, Shoulder, and Hand (DASH) score of patients with primary musculoskeletal sarcoma around the forearm treated with AO therapy, compared to that of patients treated with conventional wide resection., Methods: Subjects were 18 patients with primary musculoskeletal sarcoma around the forearm and treated with AO therapy (AO: n = 8) after marginal or intralesional resection, or conventional wide resection followed by limb reconstruction surgery (WR: n = 10)., Results: Mean age of the 18 patients was 45 years, and mean durations of follow-up for Groups AO and WR were 67 and 74.1 months. Local recurrence rates for AO and WR were 12.5% and 20% (P = 0.63), DASH disability scores were 3.9 and 21 (P = 0.04), and 5-year survival rates were 100% and 90% (P = 0.40), respectively., Conclusions: AO therapy offers maintenance of excellent upper limb function and inhibition of local tumor recurrence, representing a useful modality for limb salvage surgery in patients with sarcoma around the forearm., (2010 Wiley-Liss, Inc.)

Rhabdomyosarcoma is a common malignant soft tissue that frequently involves bone and major neurovascular structures and resection of deep-seated rhabdomyosarcoma can cause severe dysfunction in the affected limbs. Based on the mouse osteosarcoma model, we developed a new surgical approach involving photodynamic surgery (PDS), photodynamic therapy (PDT) and radiodynamic therapy (RDT) using acridine orange (AO). Six rhabdomyosarcoma cases were treated using this new modality after confirming the effectiveness of AO-PDT on human rhabdomyosarcoma cell lines. All patients had almost normal limb function after surgery, with only one recurrence. Based on these results, AO-PDS, PDT and RDT can be used to preserve excellent limb function in patients with rhabdomyosarcoma involving major nerves and vessels or bones.

Background: To maintain excellent limb function after tumor resection in patients with high-grade malignant sarcomas, we developed and established a new surgical adjuvant therapy using acridine orange (AO) after intra-lesional or marginal resection while sparing normal tissues of major nerves, vessels or bones adjacent to the tumor., Method: Our AO therapy procedure was combined with photodynamic surgery (PDS), photodynamic therapy (PDT) and radiodynamic therapy (RDT). In this study, 26 patients with primary high-grade soft tissue sarcomas were treated with AO therapy., Result: Results showed a low local recurrence rate (7.7%) and good local recurrence-free rate (88%) after AO therapy. Limb function of all patients was maintained at 100% of ISOLS criteria., Conclusion: Based on these results, we concluded that AO therapy is useful for local control after margin-positive tumor resection and for preserving excellent limb function in patients with high-grade soft tissue sarcomas., ((c) 2008 Wiley-Liss, Inc.)

We recently established a unique therapeutic modality for musculoskeletal sarcomas, combining acridine orange (AO) with photodynamic surgery (PDS), photodynamic therapy (PDT) and radiodynamic therapy (RDT); excellent results were obtained in the inhibition of local tumor recurrence after intralesional excision. However, AO is not yet approved for clinical use and intravenous injection. Therefore, methylene blue (MB), which has a very similar chemical structure to AO and is already in clinical use for other diseases, was investigated. In vitro studies using mouse osteosarcoma (LM8) cells revealed that MB-PDT had a strong cytocidal effect and that MB was not radiosensitive, showing no effect in RDT. In vivo studies showed that MB did not specifically accumulate in mouse osteosarcoma tissue and that it did not inhibit tumor growth. MB is not a better photosensitizer than AO in PDS, PDT and RDT for osteosarcoma.

A photodynamic therapy technique with acridine orange (AO-PDT) was experimentally developed and applied clinically to musculoskeletal sarcoma patients to reduce the surgical margin and obtain good limb function. Furthermore, various modalities to enhance and strengthen the cytocidal effect of AO-PDT were investigated. A recent report revealed that the use of stronger unfiltered xenon light in AO-PDT enhanced the cytocidal efficacy of this treatment modality. Therefore, in this study, we investigated whether the use of a flash wave light (FWL) from a xenon lamp, as compared to that of the conventional continuous wave light (CWL), might enhance the cytocidal effect of AO-PDT, using the mouse osteosarcoma cell line, LM8. For an equal energy dose (79.6 joules/cm2), AO-PDT using FWL (10 minutes excitation) was found to exert a significantly stronger cytocidal effect than that using CWL (18 seconds excitation). For the same excitation time (10 minutes' excitation), the use of FWL (79.6 jouleslcm2) was associated with a significantly stronger cytocidal effect of AO-PDT than that of CWL (3,820 joules/cm2). These results reveal that the use of FWL entails the need for a lower excitation energy and shorter excitation time than that of CWL for the cytocidal effect of AO-PDT to be observed against the osteosarcoma cells. In addition, FWL also has the advantage of generating low heat and of having the ability to homogenously illuminate a wider area. We therefore concluded that FWL is more useful for AO-PDT than CWL in terms of saving on the excitation time and of obtaining good efficacy of destruction of the residual tumor in the treatment of musculoskeletal sarcomas.

Acridine orange (AO) was extracted as a dye from coal tar over a hundred years ago. It has various unique biological activities and has been shown to be a useful fluorescent dye specific for DNA and RNA, a pH indicator, photosensitizer, antitumor and antimalarial drug, and detector of bacteria and parasites. It has recently been found that AO accumulates in musculoskeletal sarcomas and that after illumination of the tumors with visible light or irradiation with low-dose X-rays, the dye rapidly exerts selective cytocidal effect against the sarcoma cells. Therefore, surgery combined with photo- (PDT) or radiodynamic therapy (RDT) with AO (AO-PDT and -RDT) has been applied to human musculoskeletal sarcomas. The results of a clinical study on the outcome of this therapeutic strategy revealed that it yielded better local control and remarkably better limb function than wide resectional surgery. Based on our experimental studies, it was clarified that AO accumulates in acidic organelles or structures, especially lysosomes, depending on the acidity. An enormous number of protons are produced in cancer from lactate or CO2 under hypoxic conditions, which are moved into the extracellular fluid or lysosomes to maintain the intracellularfluid pH. Therefore, AO shows marked accumulation in the acidic lysosomes of cancer cells. Photon energy from visible light or X-rays excites the AO accumulated in lysosomes; the excited AO emits fluorescence and forms activated oxygen from intra-cytoplasmic oxygen. The activated oxygen destroys lysosomes, with the released lysosomal enzymes causing rapid death of the cancer cells. On the other hand, normal cells can exclude AO quickly because they are not acidic. Thus, AO-PDT and AO-RDT exhibit strong and selective cytocidal effect against malignant tumors. In conclusion, we believe that AO-PDT and AO-RDT exhibit selective anticancer cell activity and that AO excited by photon energy has excellent potential as an anticancer agent.

Background: Intra-operative photodynamic therapy has been applied with acridine orange (AO-PDT) to human musculoskeletal sarcomas for the past 4 years, resulting in a low local recurrence rate, within 10%, after intra-marginal tumor resection and excellent limb function. However, it is still unclear why acridine orange (AO) specifically accumulates in tumor cells, especially in malignant tumor cells. The purpose of this study was to clarify the mechanism of AO accumulation in malignant musculoskeletal tumors., Materials and Methods: Sixty-two musculoskeletal tumors, including 35 malignant and 27 benign tumors, were studied. Using freshly resected tumor material, the extracellular pH (pHe) was measured and the fluorescence intensity of AO accumulated in the tumors was measured by an image analyzer after ex vivo exposure to 1.0 microg/ml AO, followed by blue excitation. In the in vitro study, bafilomycin A1 was exposed to LM 8 mouse osteosarcoma cells, in order to inhibit V-ATPase, subsequently causing a decrease in the pHgradient (deltapH) between the intracellular pH (pHi) and extracellular pH (pHe) or between the pHi and the pHe. AO accumulation and the cytocidal effect of AO were evaluated., Results: The results of the in vivo study, using human materials freshly resected at surgery, revealed that the pHe of the malignant musculoskeletal tumors was significantly lower than that of the benign tumors and normal muscles or adipose tissues and also showed that the AO fluorescence intensity of the malignant musculoskeletal tumors was significantly stronger than that of the benign tumors and normal muscles or adipose tissues. The results also revealed that the AO fluorescence intensity negatively correlated with the pHe in tumors and normal tissues. The in vitro study showed that bafilomycin A1 inhibited the accumulation of AO in acidic organelles, such as lysosomes, and that the cytocidal effect of AO-PDT was also remarkably inhibited., Discussion: Based on results of the in vivo and in vitro studies, it is suggested that malignant musculoskeletal tumors have a large deltapH between the pHi and the pHe or between the pHi and the vaculolar pH and also that a large ApH increases AO accumulation in tumors. We, therefore, believe that AO-PDT may be more effective in highly malignant musculoskeletal tumors than low grade ones, because of their acidity., Conclusion: AO accumulation in musculoskeletal tumors was dependent on the ApH between the pHi and the pHe, or between the pHi and the vacuolar pH.

Various imaging methods have been employed for the extracorporeal detection of malignant tumors in the human body, such as scintigraphy and PET; however, none is sufficiently accurate and all are also very expensive. To resolve these issues, we attempted to develop a new imaging technique of photodynamic diagnosis (PDD) with acridine orange (AO). AO has the ability to rapidly and specifically accumulate in malignant tumors and emit brilliant green fluorescence after blue light excitation. In this study, we investigated the feasibility of PDD utilizing the fluorovisualization effect of AO, for the extracorporeal detection of mouse osteosarcoma inoculated into the soft tissues. At 2 h after intravenous administration of 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mg/kg AO, the tumor and the surrounding normal tissues were illuminated by blue light. The visual fluorescence contrast and ratio (X) of the difference in fluorescence intensity between the tumor and the surrounding normal tissues were evaluated using a high-resolution digital camera equipped with an absorption filter. In addition, the fluorescence contrast was also detected sequentially at 0.5, 1, 2, 3, 6 and 12 h after intravenous administration of AO at 1.0 mg/kg. The results revealed that the optimal condition for clear detection of the tumor was evaluation 2 h after intravenous injection of AO at 0.1 mg/kg, because it provided the best visual contrast on the digital images, and the fluorescence intensity as well as the value of X were higher as compared to the values under other conditions of dose and timing. Based on the results of an acute toxicity study of AO, the estimated LD50 of this substance following intravenous administration was 27.30 mg/kg. In conclusion, we believe that PDD using AO administered intravenously may be feasible for the detection of human musculoskeletal sarcomas in the soft tissues at extremities, and this technique might be a less invasive, less expensive, quicker and more accurate imaging modality than other previously reported imaging methods for this purpose., (Copyright 2006 S. Karger AG, Basel.)

Synovial sarcoma (SS) is one of common malignant soft-tissue tumors and is encountered most commonly in children and young adults. It frequently involves or invades major neurovascular structures and bones, and its local recurrence rate after simple resection has been reported to be as high as up to 80%. Because major nerves and vessels, as well as an adequate amount of bone, must be preserved to restore excellent limb function in cases of SS, a surgical technique entailing a low risk of local recurrence is needed. Based on the findings of recent experimental studies conducted by us using a mouse osteosarcoma model, we developed a novel therapeutic technique for SS, consisting of reduction surgery followed by photodynamic therapy using acridine orange (AO-PDT), with or without X-ray irradiation at 5 Gy. A preliminary study revealed that low-dose X-rays also excite AO like photons. After an initial study on cell cultures, this novel technique was applied to six cases of SS. A follow-up of the subjects to determine the clinical outcome revealed that none of the cases treated by AO-PDT, including the four cases treated by additional 5 Gy irradiation and the two cases not receiving any radiation, showed any evidence of recurrence or local/systemic complications during the follow-up period of 19-51 months after the surgery. Therefore, we believe that AO-PDT with 5 Gy irradiation may be an excellent novel therapeutic modality with reduction surgery to salvage excellent limb function in SS involving major nerves and vessels or bones.

Most patients with musculoskeletal sarcoma do not recover satisfactory limb function after limb salvage surgery. To achieve satisfactory improvement of limb function, we developed a unique surgical modality of photodynamic therapy using acridine orange (AO-PDT) and clinically applied it to patients with musculoskeletal sarcomas. Ten patients with primary musculoskeletal sarcomas were enrolled in the study. Of these, 6 had primary malignant soft tissue sarcoma and 4 had primary malignant bone tumor. In the AO-PDT procedure, intralesional or partially marginal tumor excision was initially conducted and microscopic curettage of the remnant tumor, which emitted green fluorescence under blue excitation after local administration of 1microg/ml AO solution, was performed using a fluorescence surgical microscope. Subsequently, blue light illuminated there for 10 minutes. The surgical wound was closed, followed by immediate X-ray irradiation of the resected area with 5 Gy in 5 out of 10 patients to enhance the effect of AO-PDT. The follow-up of the patients ranged from 24 to 48 months. All the patients (AO-PDT alone: 5, AO-PDT with 5-Gy radiation: 5) are alive; only one patient showed local recurrence of the tumor. The recurrence rate was 10%. None of the 5 patients treated by AO-PDT with radiation developed local tumor recurrence. The limb function in all the patients, except for one, recovered to the level before surgery. None of the patients clinically showed any local or systemic complications. AO-PDT may be a promising new limb salvage modality for preservation of excellent limb function in patients with musculoskeletal sarcoma.