Intravascular Administration of Acridine Orange and Zoledronate in a Bone Metastasis Model of Breast Cancer.

BACKGROUND/AIM: This study evaluated the effect of haematogenous administration of acridine orange (AO) alone and in combination with zoledronate (ZOL) on bone metastases. MATERIALS AND METHODS: E0771 cells (1.0×105 cells/10 µl) were injected directly into the right femur of female mice. The mice were divided into five groups according to treatment (drugs and irradiation) and were reared and sacrificed after 6 weeks. Micro-computed tomography (µCT) was performed to calculate the destruction rate of the femur bone. We measured tumour weight and volume at sacrifice and performed terminal deoxynucleotidyl transferase dUTP Nick-End Labelling staining of tumours. RESULTS: At 4 weeks, the bone destruction rate was lower in the AO+ZOL group than in the radiation group. At 6 weeks, the AO+ZOL group had a lower bone destruction rate than the control and radiation groups; the ZOL group had a lower rate than the radiation group. The AO and AO+ZOL groups had suppressed tumour weight and volume compared to the control and radiation groups. The number of extraosseous apoptotic cells was higher in the AO+ZOL group than in all other groups except the AO group. CONCLUSION: In a model of local bone metastasis of breast cancer, haematogenous administration of AO reduced tumour size and more so when combined with ZOL.

Recurrence analysis of intraoperative acridine orange-photodynamic therapy for dogs with intranasal tumors.

Intraoperative acridine orange-photodynamic therapy (AO-PDT) and cribriform plate irradiation are used to treat canine intranasal tumors. The purpose of this study was to evaluate the effects of AO-PDT on intranasal tumors and the recurrence rate of tumors after this treatment. Treatments with AO-PDT were performed on 38 dogs through a narrow window of the dorsal nasal cavity. The median progression-free interval was 12 mo and recurrence was detected in 21 dogs. Based on computed tomography, recurrence in 16 dogs was biased to the following areas: lateral (n = 10), medial (n = 2), ventral (n = 0), rostral (n = 0), and caudal (n = 8). Side effects were mild and included subcutaneous emphysema and rhinitis. The median survival time was 24 mo. Although AO-PDT with cribriform irradiation is an effective treatment for intranasal tumors, AO-PDT techniques should be improved to treat the nasal cavity more uniformly and thoroughly.

Analyse de récurrence de la thérapie photodynamique peropératoire à l'acridine orange pour des chiens atteints de tumeurs intranasales. La thérapie photodynamique peropératoire à l'acridine orange (AO-PDT) et l'irradiation de la plaque cribriforme sont utilisées pour traiter les tumeurs intranasales canines. Le but de cette étude était d'évaluer les effets de l'AO-PDT sur les tumeurs intranasales et le taux de récidive des tumeurs après ce traitement. Des traitements avec AO-PDT ont été effectués sur 38 chiens à travers une fenêtre étroite de la cavité nasale dorsale. L'intervalle médian sans progression était de 12 mois et une récidive a été détectée chez 21 chiens. Sur la base de la tomodensitométrie, la récidive chez 16 chiens était biaisée dans les zones suivantes : latérale (n = 10), médiale (n = 2), ventrale (n = 0), rostrale (n = 0) et caudale (n = 8). Les effets secondaires étaient légers et comprenaient l'emphysème sous-cutané et la rhinite. La durée médiane de survie était de 24 mois. Bien que l'AO-PDT avec irradiation de la plaque cribriforme soit un traitement efficace pour les tumeurs intranasales, les techniques d'AO-PDT devraient être améliorées pour traiter la cavité nasale de manière plus uniforme et plus complète.(Traduit par Dr Serge Messier).

Impact of Acridine Orange in Patients With Local Recurrent Soft Tissue Sarcoma.

BACKGROUND/AIM: Local recurrence in soft tissue sarcoma (STS) is a risk factor of worse prognosis. Although a few studies have shown that adjuvant therapy with acridine orange (AO) is effective for local control of primary STS, there have been no reports examining its effectiveness for local recurrence. PATIENTS AND METHODS: This retrospective study included 36 patients with first local recurrence of STS. Of them, 23 patients received wide excision without AO therapy (Wide group); the other 13 patients received marginal excision with AO therapy (AO group). We compared re-recurrence rates between these two groups. RESULTS: The total re-recurrence rate was 43.5% in the Wide group and 46.2% in the AO group. There was no significant difference in local re-recurrence-free survival and overall survival between the two groups. CONCLUSION: Adjuvant AO therapy combined with a marginal excision suppresses local re-recurrence rates of individuals with local STS recurrence.

Acridine Orange Encapsulated Mesoporous Manganese Dioxide Nanoparticles to Enhance Radiotherapy.

Manganese dioxide (MnO2) nanoparticles are a promising type of radiosensitizer for they can catalyze H2O2 decomposition to produce O2. Combining MnO2 nanoparticles with conventional, small molecule radiosensitizers would further enhance radiotherapy (RT) efficacy due to complementary mechanisms of action. However, solid MnO2 nanoparticles are suboptimal at drug loading, limiting the related progress. Herein we report a facile method to synthesize mesoporous MnO2 (mMnO2) nanoparticles, which can efficiently encapsulate small molecule therapeutics. In particular, we found that acridine orange (AO), a small molecule radiosensitizer, can be loaded onto mMnO2 nanoparticles at very high efficiency and released to the surroundings in a controlled fashion. We show that mMnO2 nanoparticles can efficiently produce O2 inside cells. This, together with AO-induced DNA damage, significantly enhances RT outcomes, which was validated both in vitro and in vivo. Meanwhile, mMnO2 nanoparticles slowly degrade in acidic environments to release Mn2+, providing a facile way to keep track of the nanoparticles through magnetic resonance imaging (MRI). Overall, our studies suggest mMnO2 as a promising nanoplatform that can be exploited to produce composite radiosensitizers for RT.

Impact of Acridine Orange in Patients With Soft Tissue Sarcoma Treated With Marginal Resection.

BACKGROUND/AIM: Although few studies have shown the effectiveness of adjuvant therapy with acridine orange (AO) for soft tissue sarcoma (STS) patients, no study has investigated this among cases with marginal resection. The aim of the study was to evaluate the effectiveness of AO therapy directly by comparing it to marginal resection cases that did not receive AO. PATIENTS AND METHODS: This retrospective study included 19 and 33 patients with STS who received AO therapy (AO group) and marginal resection without AO therapy (non-AO group), respectively. The patients' clinical information was collected, and the clinical courses were compared. RESULTS: The local recurrence rate in the AO group was significantly lower than that in the non-AO group (p<0.05). The local recurrence-free survival curves significantly differed between the two groups (p<0.05). High grade malignancy and no treatment with AO were identified as risk factors for local recurrence (p<0.05). CONCLUSION: AO therapy strongly suppressed local recurrence after marginal resection of STS.

Clinical Trial of Radiotherapy After Intravenous Injection of Acridine Orange for Patients with Cancer.

AIM: We previously found that low-dose X-ray treatment after systemic administration of acridine orange (AO), which is known to have a low toxicity in animals, inhibited tumor growth in experimental studies using mouse osteosarcoma. In this pilot study, we planned to verify the toxicity of intravenous injection of low-dose AO in humans and investigate the anticancer effect of radiation after systemic AO administration (iAOR) for human cancer. PATIENTS AND METHODS: Eight patients with terminal cancer were treated with iAOR. RESULTS: None of the patients exhibited an adverse effect from AO injection. Three out of the five patients who received a full course of iAOR exhibited clinical or image-based responses, whereas two patients did not. CONCLUSION: The systemic administration of AO was confirmed not to be toxic in humans, and iAOR was suggested to be potentially effective against radioresistant cancer.

Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors.

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466 nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.

Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA.

A new family of 99mTc(I)- tricarbonyl complexes and 125I-heteroaromatic compounds bearing an acridine orange (AO) DNA targeting unit was evaluated for Auger therapy. Characterization of the DNA interaction, performed with the non-radioactive Re and 127I congeners, confirmed that all compounds act as DNA intercalators. Both classes of compounds induce double strand breaks (DSB) in plasmid DNA but the extent of DNA damage is strongly dependent on the linker between the Auger emitter (99mTc or 125I) and the AO moiety. The in vitro evaluation was complemented with molecular docking studies and Monte Carlo simulations of the energy deposited at the nanometric scale, which corroborated the experimental data. Two of the tested compounds, 125I-C5 and 99mTc-C3, place the corresponding radionuclide at similar distances to DNA and produce comparable DSB yields in plasmid and cellular DNA. These results provide the first evidence that 99mTc can induce DNA damage with similar efficiency to that of 125I, when both are positioned at comparable distances to the double helix. Furthermore, the high nuclear retention of 99mTc-C3 in tumoral cells suggests that 99mTc-labelled AO derivatives are more promising for the design of Auger-emitting radiopharmaceuticals than the 125I-labelled congeners.

Intraoperative acridine orange photodynamic therapy and cribriform electron-beam irradiation for canine intranasal tumors: A pilot study.

Untreated canine intranasal tumors carry a poor prognosis. We retrospectively evaluated the efficacy of marginal tumor resection in combination with intraoperative acridine orange (AO) photodynamic therapy (PDT) and 1 fraction of 5 Gy megavoltage irradiation for canine intranasal malignant tumors. When cribriform plate invasion or turbinate destruction around the cribriform plate was present, an additional fraction of 20 Gy was delivered with an electron beam during surgery. The study included 6 dogs, 2 of which were classified as stage I, 1 as stage II, and 3 as stage IV. The median local disease-free survival time and overall survival after the treatment were 8.5 and 13 months, respectively. Recurrence was noted in 2 of the 6 dogs after 4 and 7 months. Adverse events were mild (subcutaneous emphysema in 1 case, and rhinitis in 3 cases). Combination AO therapy may increase the tumor control time of dogs with marginally resectable intranasal malignant tumors.

Pour des tumeurs intra-nasales malignes, une thérapie photodynamique administrant de l'acridine orange pendant l'opération et une irradiation par mégavoltage aux plaques cribriforms: l'etude préliminaire. Le pronostic des tumeurs intra-nasales canines non traitées est défavorable. Cette étude avait pour objectif d'évaluer rétrospectivement l'efficacité de la résection marginale d'une tumeur associée à une thérapie photodynamique (TPD) administrant de l'acridine orange (AO) pendant l'opération et à 1 fraction de 5 Gy d'irradiation par mégavoltage dans le traitement des tumeurs intra-nasales malignes. En cas d'invasion des plaques cribriformes et/ou de présence de cornets autour des lésions cribriformes, une fraction supplémentaire de 20 Gy a été administrée pendant l'opération par faisceaux d'électrons. Six chiens ont été inclus dans l'étude. Deux chiens présentaient des tumeurs de stade I, un de stade II et trois de stade IV. La durée moyenne de survie sans récidive locale et de survie globale après le traitement étaient respectivement de 8,5 et 13 mois. Une nouvelle tumeur est apparue chez deux des six chiens, respectivement 4 et 7 mois après le traitement. Les effets indésirables étaient bénins (un cas d'emphysème sous-cutané et trois cas de rhinite. L'association de la thérapie par AO améliorerait la durée de contrôle de la tumeur chez les chiens présentant des tumeurs intra-nasales malignes marginalement résécables.(Traduit par les auteurs).

Can a less radical surgery using photodynamic therapy with acridine orange be equal to a wide-margin resection?

BACKGROUND: Wide-margin resections are an accepted method for treating soft tissue sarcoma. However, a wide-margin resection sometimes impairs function because of the lack of normal tissue. To preserve the normal tissue surrounding a tumor, we developed a less radical (ie, without a wide margin) surgical procedure using adjunctive photodynamic therapy and acridine orange for treating soft tissue sarcoma. However, whether this less radical surgical approach increases or decreases survival or whether it increases the risk of local recurrence remains uncertain. QUESTIONS/PURPOSES: We determined the survival, local recurrence, and limb function outcomes in patients treated with a less radical approach and adjunctive acridine orange therapy compared with those who underwent a conventional wide-margin resection. METHODS: We treated 170 patients with high-grade soft tissue sarcoma between 1999 and 2009. Fifty-one of these patients underwent acridine orange therapy. The remaining 119 patients underwent a conventional wide-margin resection for limb salvage surgery. We recorded the survival, local recurrence, and functional score (International Society of Limb Salvage [ISOLS]) score) for all the patients. RESULTS: The 10-year overall survival rates in the acridine orange therapy group and the conventional surgery group were 68% and 63%, respectively. The 10-year local recurrence rate was 29% for each group. The 5-year local recurrence rates for Stages II, III, and IV were 8%, 36%, and 40%, respectively, for the acridine orange group and 13%, 27%, and 33%, respectively, for the conventional surgery group. The average ISOLS score was 93% for the acridine orange group and 83% for the conventional therapy group. CONCLUSION: Acridine orange therapy has the potential to preserve limb function without increasing the rate of local recurrence. This therapy may be useful for eliminating tumor cells with minimal damage to the normal tissue in patients with soft tissue sarcoma. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of the levels of evidence.

[Encounter of cancer cells with bone. Development of cancer therapy targeted on acidic microenvironment and acidic organelle of cancer cells].

Recent study revealed that most of cancer cells form acidic environment and have many, large size acidic organelle, especially lysosomes due to cancer specific proton dynamics induced by active aerobic glycolysis without TCA cycle in the mitochondoria. We have developed a new cancer therapy with acridine orange photodynamics which targeted on such cancer acidity and treated patients with malignant bone and soft tissue tumors. In this paper, we describe mechanism and clinical outcome of the therapy.

Clinical outcomes of minimally invasive surgery using acridine orange for musculoskeletal sarcomas around the forearm, compared with conventional limb salvage surgery after wide resection.

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.

Photodynamic therapy with acridine orange in musculoskeletal sarcomas.

Limb salvage involving wide resection and reconstruction is now well established for managing musculoskeletal sarcomas. However, involvement of major nerves and vessels with a large volume of muscle and skin may result in a useless limb, contributing to depression and a low quality of life. We have been studying alternative treatments for musculoskeletal sarcoma since 1990, and have recently established a regime using photodynamic surgery with cells labelled with acridine orange, photodynamic therapy with cells treated similarly and radiodynamic treatment using the effect of X-rays on such cells. These techniques have been used after marginal or intralesional resection of tumours since 1999 and have enabled maintenance of excellent limb function in patients with sarcomas.

A new therapeutic modality involving acridine orange excitation by photon energy used during reduction surgery for rhabdomyosarcomas.

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.

Methylene blue in place of acridine orange as a photosensitizer in photodynamic therapy of osteosarcoma.

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 new limb salvage surgery in cases of high-grade soft tissue sarcoma using photodynamic surgery, followed by photo- and radiodynamic therapy with acridine orange.

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.

Review. Acridine orange could be an innovative anticancer agent under photon energy.

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.

Clinical trial of photodynamic therapy using acridine orange with/without low dose radiation as new limb salvage modality in musculoskeletal sarcomas.

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.

Clinical outcome of a novel photodynamic therapy technique using acridine orange for synovial sarcomas.

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.