Outcome measures following Sono and Photodynamic Therapy – A Case Series

Sono and Photodynamic Therapy (SPDT) is a novel therapeutic modality that utilises a non-toxic photosensitive agent with reported ultrasoundactivated properties. SPDT has previously demonstrated significant tumour cell inhibition in animal studies. There has been much research into the efficacy of photodynamic therapy and development in understanding of the underlying mechanism of tumour cytotoxicity. Synergistic ultrasound activation represents a promising development to Photodynamic Therapy, as photo-activation is limited by access and penetrance issues. Ultrasound has been demonstrated to activate a number of sono-sensitive agents allowing the possibility of noninvasive targeted treatment of deeper tumour sites than is currently achievable with photodynamic therapy. This case series of 17 consecutive patients with a variety of cancer diagnoses outlines clinical outcomes over a four-year period of SPDT. The results have been encouraging in that all cases who carried our Circulating Tumour Cell Tests before and after SPDT showed a significant drop in tumour cells post-SPDT. SPDT is worthy of further investigation as an effective and well tolerated treatment for a wide variety of primary and metastatic tumours, including those refractory to Chemotherapy.


Introduction
A case series of 17 consecutive patients with a variety of Cancer diagnoses outlines clinical outcomes over a four-year period of Sono and Photodynamic Therapy (SPDT). This is a novel therapeutic modality that utilises a nontoxic photosensitive agent with reported ultrasound-activated properties. This treatment centres around the development of a specific light and ultrasound activated sensitiser (Sonnelux1) which has previously demonstrated tumour cell inhibition in animal studies and provides a new method of inducing targeted tumour cell necrosis. Many of the patients included in this case series have advanced metastatic cancer diagnoses, and most have failed to respond to conventional management approaches. We have previously published in this area 1,2 Many of these cases showed significant extension of predicted median survival and also of outcome measures as measured by Circulating Tumour Cell Tests 3 . Other Authors have demonstrated significant results using SPDT 4-6 .

Background Photodynamic Therapy -Light Activated Therapy
Photodynamic Therapy (PDT) is an established therapeutic option for a variety of pre-cancerous and malignant pathologies. The majority of PDT photosensitive agents possess a heterocyclic ring structure similar to that of chlorophyll or the haem group in haemoglobin that can be administered via topical or systemic routes. The photosensitiser becomes activated by light energy applied from an LED or coherent laser emission source. Following absorption of light at a specific wavelength by the photosensitiser, a transfer and translation occurs of light energy into a chemical reaction. In the presence of molecular oxygen this produces singlet oxygen (1 O2) or superoxide (O2 -), and induces cell damage through direct and indirect cytotoxicity 7 . A variety of photosensitisers demonstrates elective absorption into malignant cells, increasing the potential to target cytotoxicity 7,8 and limit unwanted side-effects.
Photo-activation is however limited to surface pathology, or tumour mass capable of being targeted via endoscopic access. This is due to absorption of light into surrounding tissue, which creates limitation on penetrance and the depth of photosensitiser activation. The use of new photosensitisers sensitive to longer wavelengths of light increases depth of penetration 7 , but effective non-invasive treatment of deep tumour sites remains problematic.

Sonodynamic Therapy -Ultrasound Activated Therapy
Ultrasound is a mechanical wave with periodic vibrations of particles in a continuous, elastic medium at frequencies equal to or greater than 20 kHz . It is not only perceived as safe, but has excellent tissue penetrating ability without major attenuation of its energy 8,9 . Therefore, the potential medical application of ultrasound has been evaluated extensively and has led to the routine use of ultrasound for diagnostic imaging of soft tissue 9 .
Ultrasound Activated therapy (sonodynamic therapy), the ultrasound dependent enhancement of cytotoxic activities of certain compounds (sonosensitisers), is an attractive modality for cancer treatment with potential to focus the ultrasound energy on tumour sites buried deep in tissues and to locally activate a preloaded sonosensitiser. The effect can be localised by focusing the ultrasound on a defined region and choosing compounds with tumour affinity 10,11,12,13 , causing enhanced cytotoxicity at pathological sites with minimal damage to peripheral healthy tissue. Potentiated cytotoxicity by ultrasound was first demonstrated when mouse leukaemia L 1210 cells were exposed to continuous wave ultrasound (2 M Hz, 10 W/ cm2) while suspended in nitrogen mustard solution in vitro. Mice subsequently inoculated with these cells had longer survival times than control animals that received cells exposed to the drug but not ultrasound 14 .
Following this, the application of low-level ultrasound to a biological target was found to potentiate chemotherapeutic cell killing with adriamycin and diaziquone 15 . In vivo, this combined drug and ultrasound treatment resulted in statistically significant reductions in tumour volume of uterine cervical squamous cell carcinoma implanted in the cheek pouch of the Syrian hamster compared to the chemotherapeutic alone. The ultrasound applied without the chemotherapy agent was non-cytotoxic and produced negligible temperature elevation. The photodynamic sensitisers have also been studied for ultrasound-activated properties. They have the benefit of being non-toxic unless activated and have been demonstrated to have tumour localizing properties. Hematoporphyrin, a commonly used photo-sensitiser enhanced the killing of mouse sarcoma and rat ascites 130 tumour cells exposed in vitro to ultrasound (1.92 MHz) at intensities of 1.27 and 3.18 w/cm2, from 30% and 50% to 99% to 95% respectively [16]. Possible cytotoxic mechanisms include generation of sonosensitiser-derived radicals which initiate chain peroxidation of membrane lipids via peroxyl and/or alkoxyl radicals, the physical de stabilization of the cell membrane by the sonosensitizer thereby rendering the cell more susceptible to shear forces and cavitation effects or ultrasound enhanced drug transport across the cell membrane (sonoporation) 13,17,18 .

Sonnelux-1 -A Dual Activation Agent
Light and Ultrasound Activation Sonnelux-1 is a metallochlorin complex, containing a highly purified mixture of several chlorophyllins, each with a different side chain and an average molecular weight of 942. Sonnelux-1 has photo-activation properties and has also been demonstrated to be extremely sensitive to ultrasound 16 . Safety studies, including LC50 studies of S onnelux-1 as determined in zebrafish, reveal that Sonnelux-1 is essentially non-toxic. No zebrafish death is noted at the maximum soluble concentration of the sonosensitiser (data pending publication). 7 Sonnelux-1 is registered as non-hazardous according to OSHA standards and EU directives.

Sonnelux-1 Animal Studies Demonstrating Dose Dependent Ultrasound Activated Tumour Cytotoxicity
Sonnelux-1 has demonstrated significant tumour cell cytotoxicity following ultrasound-activation using a mouse S-180 Sarcoma model 20 . Following treatment, tumour volume was monitored. Significant tumour growth inhibition was seen in the group that was administered both ultrasound and Sonnelux-1 with significant (p<0.01) reduction in mean tumour we might (see Fig. 2). No significant difference occurred with ultrasound or Sonnelux administration alone.
Significantly, cytotoxicity increased in a dose-dependent manner from 0.3W/cm2 to 1.2W/cm2 (see Fig. 3 and Fig.  4). Histology showed coagulated necrosis or metamorphic tissue which started within 2 hours of ultrasound activation 20 . Tumour cytotoxicity was also reported when a bone barrier was placed between the ultrasound exposure source and the animals under study 20 . Studies have previously supported propagation of ultrasound through bone structure 21 , and this provides further support for the possibility that sufficient ultrasound activation can be a chieved for tumour sites distant or within bone structure.

SPDT Protocol
Sonnelux-1 is administered slowly over 2 to 5 hours sublingually to provide sustained low plasma concentration. Our regulators asked us 15 years ago to use sublingual administration, we found this to be entirely satisfactory and have not had to move on to intravenous administration. Forty-eight hours after sublingual administration the patient is exposed to a light bed containing 48 panels of LEDs emitting a combination of visible and infra-red light at the frequencies 660nm and 940nm (+/-30nm). No photosensitivity from normal ambient light, artificial or natural has been noted but as a precaution, patients are advised not to stay in direct sunlight for periods over half an hour for one week following Sonnelux-1 administration. Light bed exposure time varies with shorter exposure duration in cases with larger tumour load. Ultrasound is then applied at 1W/cm2 and a frequency of 1MHz at sites of known malignant disease, with time titrated on a case by case basis. Light and ultrasound activation is repeated on three consecutive days, and the same process of Sonnelux-1 administration followed by light and ultrasound exposure   is repeated after one week to complete a treatment cycle. Ozone Autohaemotherapy is administered immediately before light bed exposure, aiming to increase P02 at the tumour site. Clinically, this has been observed to significantly increase the tumour cytotoxic effect of SPDT. A course of oral Dexamethasone is administered to patients dependent on tumour type, background physical status and total tumour volume. Alongside SPDT protocol, patients underwent supportive nutritional supplementation determined on a case by case basis.

Data Collection
Details were collated of 17 consecutive patients who received SPDT including hospital diagnosis. All of the patients except one had a Circulating Tumour Cell Test before SPDT and one after. Results have been tabulated for comparison. (Table 1) Every patient signed an Informed Consent allowing us to use their dated in an anonymous way as we are doing in this publication.

Results
All patient data is anonymously displayed in Table 1. Patient data has only been presented when a predicted median survival was known. Of those patients still alive, only those who have exceeded the predicted survival data are relevant, many patients however, are alive at the time of writing, therefore the survival benefit is unknown at the time of writing. All cases showed a reduction in Circulating Tumour Cells following SPDT except for Case 1, who refused the test.
PDT will destroy tumour down to 2cm, SDT will deal with deep-seated tumour. Therefore, combining the two deals with the tumour load and the distribution of the tumour in our patients. In the past we have tried using Light Bed treatment alone, this has some clinical benefit but this is maximised by adding in SDT.

Discussion
SPDT using Sonnelux has shown significant promise over a fifteen-year period as a safe and well tolerated noninvasive treatment, even in advanced metastatic cancer. Extension and median survival times are here reported, with patients of various cancer diagnosis.
Second and subsequent courses of Sono and Photodynamic Therapy may have further benefit in reducing tumour mass and inhibiting tumour cell growth without the total dose limitations of radiotherapy. There is a trend in the cases reported here, that further treatment reduced significantly on previous circulating tumour cell numbers.
It is suggested that unlike immunologically silent genotoxic damage produced by radiotherapy and chemotherapy, photooxidative cytotoxic lesions generated by Sono and Photodynamic Therapy are extra nuclear and result in a rapid cell death as it alerts the host's innate immune system 22. Neutrophil mobilisation and innate immune cell activation are responsible for the development of tumour antigen-specific adaptive immune cascades that contribute to the eradication of Sono and Photodynamic Therapy treated cancers. This is further supported by in vitro studies which establish the tumour cells treated by PDT can be used for generating potent vaccines against cancers of the same origin 23 .
Tumour hypoxia has been found to a characteristic feature in many solid tumours 24 . It has been demonstrated the tumour hypoxia, either pre-existing or as a result of oxygen depletion during photodynamic therapy can significantly reduce the effectiveness of PDT-induced cell killing.
This study reports that when Sono and Photodynamic Therapy is combined with hyperoxygenation the hypoxic condition could be improved and the cell killing rate at various time points after Sono and Photodynamic Therapy could be significantly enhanced 25 .
Previously, it has been shown in arteriopathic patients that Ozone Autohaemotherapy has a therapeutic potential by increasing oxygen delivery in hypoxic tissue 26 .
Clinically, it appears that greater tumour response is Sono and Photodynamic Therapy following Ozone Autohaemotherapy. This would seem to relate to an increase on singlet oxygen levels in the tumour microenvironment.

Conclusion
The limitations of this study are that it is observational, and therefore this supports the suggestion for further studies using this particular approach. Sono and Photodynamic Therapy warrants further investigations, a non-invasive, well tolerated, clinically effective targeted cancer treatment capable of tumour cell necrosis and both superficial in deep malignant sites. All of the patients reported here showed a drop pre and post treatment with SPDT of their circulating tumour cells. This is indeed a remarkable finding.

Conflicts of Interest
There are no conflicts of interest.