DOVE CLINIC REPORT
OBSERVATIONAL OUTCOMES ON ALL PATIENTS TREATED IN A 12 MONTH PERIOD WITH SYSTEMIC PHOTODYNAMIC AND SONODY­NAMIC THERAPY (SPDT/SDT) USING A TIN CHLOROPHYLLIN BASED SENSITIZER
By Dr Julian Kenyon,
Medical Director
Dove Clinic,
February, 2007
Summary
Patient results reported here are very encouraging. In the majority of patients, biochemistry pre- and post­SPDT/SDT shows that there has been significant tumour cell destruction. This includes the destruction of tu­mours deep in the body.
Almost all patients with bone secondaries develop increased pain post SPDT.
A clinic based in Australia (Opal Clinic, ed) also using SPDT/SDT has used photodynamic diagnosis to observe bony metastases. With this diaglostic technique, a laser light is used to stimulate fluorescent radiation from the sensitizer in tumours. The technique is only applicable to tumours relatively close to the skin. They treated a patient with non small cell lung cancer with bony metastases in the lumbar 4 vertebra. Photodynamic diag­nosis showed that these tumours disappeared after SPDT/SDT therapy. The patient also reported that he felt " 50 % better° three months after starting SPDT/SDT, and was still well after 5 months. We are looking into the use of this diagnostic method In order to look at the bone secondary situation in more detail.
A small number of patients have had a complete response, it is too early to say if their tumours will recur.
It would seem that a sensible woy forward with patients with significant tumour load, is to repeat this form of photodynamic therapy as necessary to continue tumour destruction and to cope with any recurrences.
We are continuing to audit these cases in as detailed a way as possible.
Introduction
We use a tin chlorophyllin based agent which is sensitive to red light (636 nanometres) and to ultra-
sound. Animal studies using the mouse sarcoma 180 model, show that ultrasound ( done in complete dark­ness) destroys these tumours. (X. Wang, M.D, Friendship Hospital, Guangzhou, China, personal communica­tion).
Where there is significant tumour mass, we have to control the inflammatory response which occurs following SPDT. Practically all tumours swell initially if the SPDT has been successful, due to a release of large concen­trations of pro inflammatory cytokines. The most effective way of controlling this is to use Dexamethasone at a varying dose depending on the severity of the symptoms post SPDT in each particular patient.
We have begun to fractionate treatments, as a result of our clinical experience with patients with significant tumour mass.
Judging results from photodynamic therapy can be challenging as when initially the tumour swells, the tumour looks bigger on scanning. Because private scans are relatively expensive, doing scans before and after each course of SPDT is not an every day option for us. Therefore, we tried to look at biochemistry pre and post SPDT. We have looked at tumour markers, relevant to the particular patient being treated and also the tests tumour marker 2 pyruvate kinase and cell free DNA. We have compared test results to controls in patients with stable cancer. Broadly speaking, if we get a significant change in either marker post SPDT, this corre­sponds to a clinically useful response. If no significant change, then there has not been a significant response to the SPDT.
The vast majority of our patients are late stage cancer patients who have previously had surgery, chemother­apy and radiotherapy.
The SPDT/SDT therapy was well tolerated.
Treatment
Where there is significant tumour load, there is a marked release of pro-inflammatory cytokines. The resulting inflammatory response has, in some cases, to be controlled using an appropriate dose of Dexamethasone. Whenever possible we carried out, pre and post SPDT, standard blood tests, cell free DNA, tumour marker 2.

pyruvate kinase and an appropriate tumour marker, depending on the case. The laboratories measuring cell free DNA and pyruvate kinase have, in each case, one specific biochemist dealing with these tests. When these biochemists were away on holiday, or where one of the periods when patients were having SPOT coin­cided with public holidays such as Christmas, Bank Holidays, etc. the blood tests could not be done.
We also looked at using telomerase but found that the results obtained differed wildly both in the control sub­jects and in the SPDT patients, so this was found not to be a useful measure.
We did control tumour marker 2 pyruvate kinase, cell free DNA and standard tumour markers on a range of stable cancer patients. We are currently having these results statistically evaluated. When these results are available we will add P values (probability value) to this report.
Tumour Marker 2 Pyruvate Kinase
Cell proliferation is a process that consumes large amounts of energy. A key sensor for this regulation is the glycolytic enzyme, pyruvate kinase, which determines whether glucose carbon is channeled to synthetic proc­esses or used for glycolytic energy production. The mammalian tumour marker 2 pyruvate kinase isoenzyme, can switch between a less active dimeric form and a highly active tetrameric form which regulates the chan­neling of glucose carbons either to synthetic processes (dimeric form) or to glycolytic energy production (tetrameric form). Tumour cells are usually characterized by a high amount of the dimeric form leading to a strong accumulation of all glycolytic phosphometabolites above pyruvate kinase. Therefore this test measures glycolytic activity in the body. Tumours tend to be glycolytic.
Essentially, what we have found in our observational study is that pyruvate kinase may go up or down in any particular patient but any significant movement usually corresponds to a clinically useful response.
Standard Tumour Markers
Standard tumour markers, such as CA 125 (ovary), CA (oesophagus, lung, bile duct, pancreas, bladder,colon), CA 19.9 (oesophagus, bile duct, pancreas) CA 15.3 (breast) and prostate specific antigen (prostate), usu‑
ally go up post SPDT but this is not always the case. Any significant changes up or in some cases down post SPOT correlate with a useful clinical response.
Cell Free DNA
The term `Free-DNA' is widely used, but cell-free DNA is more correct. Most of the non-related DNA in blood plasma is likely to be bound to protein molecules (2). Hence, before measuring cell-free DNA it is appropriate to use a reagent that uses a proteinase to assist in freeing DNA that is bound to proteins (3). Most circulating DNA has been released from degrading cells and is mainly present as nucleosomal elements from the enzy­matic chopping-up of the genomic DNA (4). In healthy people the circulating cell-free DNA is at a very low level (2,5). The top end of normal is 9 units. Higher concentrations are found in malignancy (6-11), autoim- mune disorders (4) and severe Infections (12,14). Burns and traumatic injuries can also show high levels of Cell-Free DNA. In other words increases are associated with significant disease (15).
The Use of Ozone Autohaemotherapy With SPDT
We obtained significantly better results by using ozone autohaemotherapy before each SPDT/SDT treat­ment. Tumour hypoxia ( low oxygen levels, ed) is often marked, and during SPOT oxygen is consumed. See Sitnik et al (16) on the reduction of tumour oxygen levels during and after photodynamic therapy. iMIMIDSISSISMIS
There is increasing evidence that killing tumour cells using photodynamic therapy resulting in tumour cell ne­crosis, also increases expression of tumour antigen. This should lead to more effective anti-tumour vac­cines. It is impossible to say at this early stage whether increased expression of tumour antigen leads to anti­gen specific T cell responses (17).
IIKUSUCSalliingaski
The main method of cell death in SPDT is by tumour cell necrosis, producing a marked increase in pro inflam­matory cytokines with hence a marked inflammatory response. This can last for several weeks. The use of Dexamethasone, in particular is especially useful in terms of controlling this reaction.
59

Appendix 1. Sonodynamic Therapy
A review of research into the uses of low level ultrasound in cancer therapy.
Tinghe Yu a,*, Zhibiao Wang a, Timothy J. Mason b
a Institute of Ultrasound Engineering in Medicine, Chongqing Medical University, Chongqing 400016, PR China
b Sonochemistry Centre, School of NES, Coventry University, Coventry CVI 5FB, UK
Received 11 May 2003; accepted 9 June 2003
Abstract
The use of low power ultrasound in therapeutic medicine is a developing field and this review will concentrate on the applications
of this technology in cancer therapy. The effects of low power ultrasound have been evaluated in terms of the biologi­cal changes
induced in the structure and function of tissue. The main fields of study have been in sonodynamic therapy, improving chemotherapy, gene therapy and apoptosis therapy. The range of ultrasonic power levels that can be effectively employed in therapy appears to be narrow and this may have hindered past research in the applications in cancer treatment.
2003 Elsevier B.V. All rights reserved.
Keywords: Cancer; Therapeutic ultrasound; Low-power ultrasound
1. Introduction
Therapeutic ultrasound is defined as the use of ultrasound for the treatment of diseased organs or structures.
This field is continuously expanding and new clinical applications are being developed constantly. Such clinical advances have been made possible by a number of factors including advances in transducer design technology, laboratory experiments to determine the precise chemical reactions taking place during or following exposure to ultrasound and advances in measurement and calibration of acoustic power for the safe application of ultrasound in therapy. Somewhat surprisingly, given some quite remarkable laboratory results, progress in the wider clinical use of ultrasound
for therapy has been very slow except in some welldefined fields such as extracorporeal lithotripsy, physiotherapy, ultrasonic surgical instruments and, more recently,
high intensity focused ultrasound (HIFU).
Throughout this review low power (or low level) ultrasound refers to the acoustic energy delivered by the
transducer. The energy may be applied over a range of frequencies that can be generally grouped into those
shown in Fig. 1 but are mostly in the so-called physiotherapy region.
Biological effects of ultrasound and their applications are a rapidly expanding research area. In recent years ultrasonic therapy for tumours has been developed successfully. HIFU has been used to treat solid tumours
and its efficacy and safety have been confirmed in clinical investigations [1]. HIFU can be carried out either as
a radical surgery or as a palliative therapy. According to some reports in the literature, some patients with inoperable liver masses are still alive and free of tumour 24
months after receiving HIFU therapy [2,3]. On the other hand, researches into the bioeffects of relatively lowintensity ultrasound on malignant tissues and their applications
are still in the process of investigation. The
63

use of this type of low-intensity ultrasound-therapy has great potential in that it can be relatively easily applied. There have been a series of investigations which appear to show that the responses of malignant cells to
low-intensity ultrasound are not identical to those of normal cells in that cancer cells were more prone to being killed [4,5]. Low-intensity ultrasound can also suppress cell proliferation and clone formation, improve the effects of anticancer chemicals and deactivate cells via indirect mechanisms [4-7]. These findings revealed
• Corresponding author. Tel.: +86-23-68485-022; fax: +86-23-68485- 023.
E-mail address: yutingher4;hotmail.com (T. Yu).
1350-4177/S - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1350-4177(03)00157-3
Ultrasonics Sonockeinistry 11 (2004) 95-103 www.elsevier.comAocate/ultsonch
that low-intensity ultrasound has distinct potential as a technique for cancer treatment.
2. Evaluating biocffects from the perspective of tissue structure and function
The bioeffects normally associated with exposure to ultrasound are heat, mechanical effects and acoustic cavitation. However, these three mechanisms do not function in the same way. Bioeffects are also intensityand frequency-dependent. A higher intensity benefits heat-production, and a lower frequency favours the occurrence of cavitation. Therefore the acoustic parameters must be selected carefully when using ultrasound
in therapy according to the objective required.
The exposure of biological tissues to ultrasound can
result in structural and/or functional alterations. Structural changes range from slight but repairable damage
to immediate death. The functional alterations include proliferation, migration, synthesis, secretion, gene expression and membranous action, etc. [4,6,8,9]. On most
occasions, structural changes in tissue brought about functional alterations and vice versa. Occasionally only functional alterations were detected in cases where the structure change was too small to be identified.
From the literature and our own work, we believe that the bioeffects of ultrasound and their applications
can be analyzed from the perspective of tissue structure and function. The sonication "level" was determined by intensity, frequency and exposure duration, etc. There are two critical levels in respect of tissue structure, one relating to the onset of cell damage (Li) and the other to
cell death (Ln). Morphological changes occur when tissues are exposed to an insonation above Li. Cells would
be immediately deactivated if the ultrasound level was PLD. Biological effects can also be understood from the perspective of tissue function. The sonication acts as either an activator or an inhibitor. Variations of structure and function with the elevation of the sonication
level are illustrated in Fig. 2. The functional change is biphasic and the structural alteration monophasic. Both structure and function are affected when tissues are exposed to levels just above Li. Therefore, it is important
to achieve a balance between structural and functional changes.
Biomedical applications of ultrasound can also be evaluated from the perspective of structure and function. We have divided these applications of ultrasound

into two groups; one to mainly induce structural alteration and the other to mostly modulate function. Levels
above LA resulted in immediate cell death, so they can be used to destroy tumours and tumour-like lesions, such as warts and benign prostate hypertrophy. These applications are included in the group that induces structural alterations. Levels around Li mainly induce functional changes; therefore they are ideal approaches to modulate tissue functions, such as gene expression
and protein synthesis. Ultrasound at this level deactivates tissues via an indirect mechanism.
In previous studies, the ultrasonic "level" was determined by the intensity and an intensity of 3 W/cm2 (or
sometimes 2 W/cm2) was regarded as the critical value between low-level and high-level ultrasound. We believed that it was more reasonable to distinguish such
levels from the bioeffects produced. This was because,
(1) applications were based upon bioeffects, (2) the intensity in vivo was affected by lots of factors (tissue type, functional status and exogenous factors, etc.). Intensities observed in situ were dramatically different despite an identical applied acoustic intensity. As a result, there are great differences in the responses in tissue, ranging from zero reaction to complete deactivation. Furthermore, therapeutic ultrasound is usually considered to operate
in the range of non-linear acoustics [10], resulting in difficulties in predicting ultrasonic behaviour in tissue. Fig. 2. Illustration of analysis of bioeffects of ultrasound and their applications from perspectives of tissue structure and function. Li: the critical level far injury; Ln: the critical level for death. (I) Therapy for tumor and tumor-like lesion; (II) gene expression, molecular delivery and fracture healing, etc. Solid curve: structure, dotted curve: function. First published in "Biological effects of ultrasound exposure on adriamycin-resistant and cisplatin-resistant human ovarian carcinoma cell lines in vitro (T. Yu et al., Ultrasonics Sonocheniistry, this issue), with permission from Elsevier B.V.
Fig. 1. General classification of the frequency ranges of ultrasound.
96 T. Yu at al. / Ultrasonics Sossochembtry 11 (2004) 95-103
We suggest that ultrasound waves, which mostly induce structural alterations, are high-level. On the other hand, those that mainly modulate tissue functions are lowlevel. Despite the fact that the use of low-level ultrasound
in therapy can be easily administered it has lagged behind the use of high-level ultrasound. We believe that
this is because it is a two edged sword, in that it has both positive and negative effects. It can suppress the mitosis of cancer cells benefiting treatment; however, it could also trigger the proliferation of malignant tissues thus contributing to spreading and metastasis. This suggests that the acoustic parameters and the exposure approaches must be determined strategically while treating malignancies with low-level ultrasound. Only in this way can the maximum therapeutic effects be realized, and the side effects minimized.
3. Sonodynamic therapy
Sonodynamic therapy (SDT) is related to photodynamic therapy (PDT), in which therapeutic effects can
be mediated by free radicals. Some chemicals produce free radicals on irradiation with light, especially laser. These active molecules destroy biological tissues, thus producing therapeutic effects and PDT has been
adopted in clinical cancer therapy. Investigators have found that ultrasound exposure can play a similar role and this has been termed SDT. SDT results from the

non-thermal effects of ultrasound, especially cavitation [11].
Ultrasonic cavitation generates free radicals from the breakdown of water molecules. The initial step in the decomposition of water is the production of hydrogen and hydroxyl radicals. Other species, such as hydrogen peroxide, singlet oxygen and superoxide ions, are formed later depending on the specific conditions [12]. Hydrogen superoxide is a very reactive molecule, which can directly deactivate large molecules, such as proteins and nucleic acid. It can also lead to the generation of other free radicals with extensive bioactivities. For these reasons, hydrogen superoxide might be thought of as an amplifier for the production of active ions. Other chemicals can be chemically activated by exposure to ultrasound, resulting in the production of a large number
of active ions. These chemicals are known as sonosensitizers. A series of in vitro and in vivo trials.
confirmed that SDT, in which either the ultrasound
or the chemical had no or very low cytotoxicity, could efficiently destroy malignant cells/tissues [13-15]. Ultrasound waves could be precisely focused on the target
volume, which made it possible to control the generation of active radicals in a definite area, so only preselected tissues were damaged. This indicated that SDT has the potential for targeted therapy.
The majority of sonosensitizers are porphyrins and their derivatives. Hematoporphyrin, pheophorbide A, photofrin, photofrin II, ATX-70 and ATX-S 10 have been used in SDT [13-20]. Other compounds, such as
merocyanine 540, erythrosin B and dimethylformamide, can also be chemically activated by sonication [21,22]. Non-steroidal anti-inflammatory drugs, tenoxicam and
piroxicam, can be used in SDT [23,24]. In vitro investigations showed that SDT led to cell lysis in erythrocytes,
sarcoma 180, L1210 and HL-60 and others
[13-15]. Animal experiments suggested that this therapy
was effective in treating sarcoma 180 and colon 26 carcinoma [15,18-20]. Umemura et al. compared the effect
of hematoporphyrin with that of ATX-70 in sarcoma 180 in vitro [16]. It was possible to double cell kill with hematoporphyrin at a concentration of 801M using ultrasound (4.5 W/cm2, 1.93 MHz) but it was improved
by a factor of 3 using ATX-70 under the same conditions. This finding suggests that the structure of the
sonosensitizer has an impact on the efficiency of SDT. Investigations into the mechanism of SDT revealed that active oxygen, especially singlet oxygen, was the
mediator in the porphyrin- and non-steroid chemicalinduced SDT. Its efficiency could be blocked by histidine,
the scavenger for singlet oxygen [16,17,19,23,24]. Investigators found that free radical scavengers and antioxidants, such as mannitol, vitamin C, vitamin E
and superoxide dismutase (SOD), could reduce cavitation- induced tissue damage [25-27]. These chemicals
could be used to protect normal tissues from being destroyed. The pharmacokinetics of porphyrin have been investigated. In order to destroy target malignant tissue
and reduce the poisoning of normal cells, insonation was administered at the specific time when the ratio of concentration of the chemical in the cancerous material to that in plasma reached a maximum. By this method,

the therapeutic effects were realized satisfactorily and side effects on normal tissues reduced [15,18]. However,
the tissue distribution of sonosensitizers was agentdependent and this gave rise to other problems:

1. The therapeutic concentration of the chemical may not be reached in a specific tissue.
2. The concentration gradient between malignant tissue and its adjacent normal tissue may not be high

enough to carry out SDT safely.

1. The concentration in the target tissue could be lower than that in plasma, resulting in hemolysis and

blood cell rupture.
We believe that endoscopic ultrasound and/or intracatheter ultrasound are probably the key, by which the
ultrasonic energy can be delivered directly to the malignant lesion. Exploring a series of sonosensitizers
with specific pharmacokinetics characteristics makes it
T. Yu et al. / Ultrasonics Sonochendstry 11 (2004) 95-103 97 possible to select a specific agent for a specific tissue or organ. This provides an effective approach for SDT.
Jin et al. reported a treatment of murine skin squamous cell carcinoma using a combination of PDT and
SDT. The medial survival period in animals receiving combination therapy (>120 day) was longer than that in mice receiving only PDT or SDT (77-95 days). Pathological examinations revealed that the combination of
SDT and PDT induced tumour necrosis more extensively [28].
Another advance in SDT was provided by the introduction of antibodies. An antibody was coupled with
the sonosensitizer, so that they could link to target cell membrane specifically and efficiently. During insonation the target cells were destroyed efficiently. This technique made therapy more precise [29]. This approach shows great promise for the improvement of antibody-directed target therapy.
4. Enhancing chemotherapy
Chemotherapy plays a very important role in cancer treatment however the application of anticancer agents is hampered by their adverse effect on normal tissues. Oncologists have focused on enhancing malignant cells destruction while at the same time reducing side effects. Unfortunately the development of drug-resistance has also contributed to the failure of some treatments. Ultrasound exposure can enhance the cytotoxicity of anticancer chemicals to cancer cells in vitro. If the same concentration of cytotoxic agents are used, more cells are killed if sonication is applied. This has made it possible to lower the dosage while maintaining or even improving the therapeutic efficiency. As a result, the patient's tolerance to chemotherapy is ameliorated. Researchers have shown that sonication can synergize the effects of adriamycin, cisplatin, 5-fluorouracil (5-FU), arabinosyl cytosine (Ara C), cisplatin, boron compound HB (dihydroxy (oxybiguanido) boron (III)
hydrochloride monohydrate), diaziquone and 4o-O-tetrahydropyranyladriamy (THP) [7,30-34]. The synergism
has been confirmed in cells with tissue types of
ovarian cancer, breast cancer, cervical cancer, leukaemia, Swiss ascites tumour and fibroblast [7,31-33].
Either continuous wave or pulsed wave (including toneburst ultrasound) can be used as the sensitizer [7,32]. We

found that the sequence of administering cytotoxic agents and sonication had an impact on the therapeutic efficiency. If adriamycin was given prior to ultrasound exposure, the cell survival rates were lower than those
obtained when insonation was performed before adriamycin administration [7].
In the in vitro experiments, cells were suspended in liquids and the mixture was then exposed to ultrasound. This induced cavitation in the liquid i.e. extracellular
cavitation. Extracellular cavitation can be detected instrumentally and is capable of rupturing cell membranes.
However, the threshold for cavitation in vivo is
much higher than in vitro. Further it is difficult to detect cavitation in vivo and to distinguish intracellular cavitation from extracellular cavitation. These factors make
the investigations of the effects of ultrasound in vivo difficult to characterise. Investigations of tumours in animals confirm that there is a synergism between anticancer drugs and ultrasound exposure in vivo. The co-administration of anticancer agents and ultrasound suppressed tumours more significantly than drugs alone and ultrasound in the absence of drugs had very limited antitumour activity. Examples of drugs which could be efficiently synergiz. ed by ultrasound exposure in vivo are adriamycin, 5-FU, BB, Ara C and bleomycin [31,33, 35,36].
Although ultrasound-induced heat was a stimulator
of membrane permeability, many investigators believe that cavitation is the mechanism of the synergism between anticancer drugs and low-level sonication. Free
radicals generated by acoustic cavitation can damage cell membranes resulting in the promotion of membrane
permeability. This improves the trans-membrane transportation of drug molecules resulting in an increase in
intracellular drug accumulation. Support for a nonthermal effect as the mechanism for the ultrasound-induced synergism comes from the absence of detectable temperature-rise in many investigations.
There were no unanimous conclusions about structural changes in the cell membrane although its permeability was increased by ultrasound. Indeed many
researchers believe that no significant structural changes occur. Saito et al. reported that ultrasound-permeated corneal cells could not be morphologically distinguished from those unaffected cells [37]. However, Tachibana
et al. found that sonication resulted in a reduction of microvilli and membranous laminar ruffles and even membrane pore formation in HL-60 cells [21]. We have investigated, by transmission electron microscopy, human ovarian carcinoma cells exposed to ultrasound which enhanced the cytotoxicity of adriamycin. Only swollen mitochondria and cytoplasmic vacuoles were detected. The cytoplasmic vacuoles are usually regarded as direct evidence of cavitation.
The interaction between ultrasound exposure and microcapsulated adriamycin has been investigated in
recent years [38-40]. The ICso observed for free adriamycin and co-polymer micelle P-105 were 1.25 and 2.25
1g/m1 respectively, which were decreased to 0.9 and 0.19 1g/m1 under sonication [38]. Similar findings were found for paramagnetic analogue ruboxyl [39], suggesting that the form of preparation affected the synergism resulting

from insonation.
The ultrasonically induced increase in intracellular drug accumulation cannot explain all the synergistic
98 T. Yu at al. / Ultrasonics Sonochanistry 11 (2004) 95-103 effects. We have evaluated the dosage-response curve of human ovarian carcinoma cell line 3A0 exposed to adriamycin and ultrasound using a radiation biology approach, because the rate of cell-kill by anticancer chemicals followed first-order kinetics [41]. The singlehit, multi-target model was used to fit the curves, and Do
and N were used to reflect the effects of ultrasound exposure (Table 1).
Cells were exposed to adriamycin only in group ADR, to adriamycin prior to sonication in group
ADR+US and to the anticancer drug following ultrasound exposure in group US +ADR. The synergism
occurred in both group US +ADR and. group
ADR+US. Survival rates in group ADR+US were lower than those in group US+ ADR. The group ADR was used as the reference for calculating the ratio. These findings suggest that exposure to ultrasound alter the intrinsic parameters of the cells, resulting in a shift of response to other stimuli. Sonication, which alone has zero or very slight cytotoxicity, lowered the threshold of cell deactivation. The results also revealed
that the method of insonation has an impact on the final effects of ultrasound. We showed that ultrasound-induced synergism also worked in human ovarian carcinoma adriamycin-resistant cells and that the reversal attributable to verapamil could be enhanced by sonication [42].
Ultrasonically induced collapse of microspheres can
be used in the control release of drugs. Thus if anticancer chemicals are encapsulated in microspheres they
can be transported to the target organ via circulation, then ultrasound can be used to induce their collapse to release the drugs. Using this technique, cytotoxic drug
molecules have been targeted on malignant lesions directly and efficiently. In such cases, the collapse of
cavitation bubbles leads not only to the release of drugs but also the permeabilization of surrounding cells/tissues (vessel endothelium, basal membrane). This could
assist in the trans-barrier transportation of the drug, such as the blood-brain barrier and blood-testes barrier (Fig. 3).
The ultrasonically induced permeability was found to be intensity and exposure-duration dependent, and the
effect was transient as long as the cell was not deactivated. Accordingly, the permeability can be adjusted to
lead to maximum beneficial effects and investigators have tried to quantify the permeability according to
acoustic parameters. Liu et al. believe that the permeability is controlled by acoustic pressure at 1/2 driving
frequency and its ultraharmonics [43]. As the bioeffects are tissue-dependent, an identical acoustic parameter resulted in various changes among different tissues. This suggested that estimating permeability based only upon ultrasonic characteristics is not an adequate approach.
5. Gene therapy
Gene therapy is regarded as a very promising technique by which malignancies could be cured radically. However, thus far no satisfactory effects have been

found in clinical trials concerning cancer treatment
using gene therapy [44].
Two problems must be solved in gene therapy: (a) the
transfer of a target DNA sequence and (b) the control of expression of the therapeutic genes transferred. Therapeutic
effects can only be attained when adequate genes
are transferred into target cells. For safety reasons, it is necessary to ensure that the target DNA will express
within a specific range and within a specific tissue/organ. In other words, the expression level of the gene must
also be kept controlled so as not to affect the normal
physiological functions of tissues once the curative effects are realized.
Ultrasound exposure can be used to improve transfection efficiency. Tata et al. transferred a plasmid encoding
GFP into prostate cancer cell line LnCap using
ultrasound. Both continuous wave (932.7 kHz, IsKrp 1.67 W/cm2) and tone-burst ultrasound (IsArA0.33 W/
cm2, LSAT? 1.67 W/cm2, 932.7 kHz in sine wave, duty cycle 20%, tone burst repetition frequency 10 Hz to 10
kHz) were adopted. Continuous wave induced a 50% transfection efficiency, and tone-burst ultrasound with a
repetition frequency of 10 Hz led to 65% transferred cells [45].
Sonication has the potential of shearing/denaturing DNA through cavitation. It is therefore necessary to
protect the DNA in order to maintain gene integrity. Investigators found that plasmid DNA is protected
against cavitation induced damage when complexed with cationic liposomes [46].
Ultrasound can modulate gene expression in vitro and in vivo. Flow cytometry revealed that ultrasound Table 1
Values of Do and N in three groups while adopting single-hit, multitarget
model
Group Do (1g/m1) Ratio N Ratio
ADR 5.8556 • 10 25.2999. 10
US+ADR 4.2061 • 10 20.7183 2.9080 • 10 10.5487 ADR+US 6.8515 10 21.1701 1.3180 • 10 10.2487 Single-hit, multi-target model: S % 1 Y21 expb N, S: survival
rate; D: dosage.
Fig. 3. Illustration of target release of anticancer agents by co-administration
of cavitation bubble collapse and microencapsulated
chemicals. (A) Microencapaulated chemical; (B) cavitation bubble.
T. Yu et al. / Ultrasonics Sonnehemistry 11 (2004) 95-103 99
makes more transfected cells express target protein [45]. Similar findings were reported by Unger et al. [47]. Ultrasound with an intensity of 0.5 W/cm2 and a frequency
of 1 MHz enhanced gene expression in Hela, NI-1/3T3
and C127I, into which DNA was introduced by liposomal transfection. Aggrecan gene expression was augmented
by ultrasound exposure in a rat femur fracture model [48]. Artificial cavitation induced by contrast
agents can increase gene transfection and its expression. This was confirmed in experiments performed by Bao
et al. [49] and Greenleaf et al. [50]. These results suggest that cavitation is the main mechanism of ultrasoundinduced
transfer and expression. The transfection efficiency attributable to ultrasound was higher than that
due to some other techniques. Encouragingly, ultrasound
could transfer into quiescent cells with the same efficiency as that of proliferating cells.
Researchers believe that the ultrasound-induced
transfection is mediated via a mechanism termed "sonoporation" [49], which was due to acoustic cavitation.

Sonoporation can be considered to be the same as the promotion of membrane-permeability induced by ultrasound. Accordingly, only transient and repairable
sonoporation can be applied to gene therapy. This indicates that ultrasound exposure should be administered
precisely. The exact mechanisms of sonication-enhanced gene expression remains unclear, although investigators believe that non-thermal effects are the cause.
A temperature rise of 5-8 i C due to focused ultrasound exposure results in an expression of HSP mRNA
in the focal region and the surrounding tissue with a index of 3-67 in rat muscle [51]. The promoter of HSP is sensitive to hyperthermia and this gives rise to a potential technique for controlling gene expression using ultrasound. Target DNA was inserted downstream of
the HSP promoter, or other promoters that were sensitive to temperature. Such a DNA segment was introduced into target cells and then the gene expression
could be modulated by altering the temperature in the tissue. Ultrasound could be used to induce the temperature rise quantitatively and precisely. This approach
could regulate not only the expression within a specific region but also the expression level.
6. Apoptosis therapy
Apoptosis, (the normal sequence of events leading to cell death), is a frequent phenomenon in malignancies, however apoptosis therapy is an effective approach for cancer treatment. Apoptosis can occur spontaneously
and be induced by many factors [52]. In cancer treatment it is induced either by radiotherapy or by chemotherapy. Malfunction of initiating apoptosis is one of
the factors which result in the failure of such therapy [53]. Sonication can trigger apoptosis in both normal and malignant cells. Ultrasound-induced cell death has been confirmed in leukemia cell lines K562, HL-60, KG1a, Nalm-6 and U937 [54-57]. Contrast agents and dissolved gases enhance ultrasound-induced apoptosis but free radical scavengers can protect against this form
Fig. 4. Apoptotic body in human ovarian carcinoma cells after ultrasound
exposure detected by transmission electron microscope.
Fig. 5. Apoptosis in human ovarian carcinoma cell line detected by 1SEL assay. Apoptotic cells were exposed to adriamycin alone (left) and combination of adriamycin and sonication (right). C when cells were treated with a combination of adriamycin and sonication.
100 T. Yu at al. / Ultrasonics Sonochensistry 11 (2004) 95-103
of apoptosis [57]. Honda et al. suggest that ultrasound initiated apoptosis occurs via the mitochondria-caspase pathway [55]. Feril et al. report that the non-thermal effect of ultrasound (1 MHz, 0.5 W/cm2) enhances the hyperthermia-induced (44 1 C for 10 min) apoptosis in
U937 cells but increasing the power to 1.0 W/cm.2 potentiates instant cell lysis [56].
We have investigated the effects of ultrasound on apoptosis in solid ovarian carcinoma. Flow cytometry revealed a sub-G1 peak after ultrasound exposure and this was confirmed by ultrastructural examination (Fig. 4). Furthermore, in situ end labelling (ISEL) showed that adriamycin-induced apoptosis was enhanced by ultrasound (Fig. 5). We have investigated this effect by evaluating the change of apoptosis ratio and that of cell survival with the elevation of adriamycin concentration. We believe that the lowering of the thresholds for both apoptosis and oncosis provide the mechanism for the synergism attributable to ultrasound exposure.

1. Other aspects Ultrasound (1.5 W/cm2, 20 kHz) can inhibit the adhesion

and migration of smooth muscle cells [81 On the
other hand, neutrophil adhesion to endothelial cells was
enhanced by therapeutic ultrasound (1.6 W/cm2, 1.0
MHz) [58]. Investigators also found that low-level ultrasound
could stimulate the synthesis and release of
cytokines [9,59]. Potential values of these bioeffects for
cancer therapy need further investigation.

1. Conclusions

Experimental investigations suggest that cancer
therapy using low-level ultrasound is a promising
technique and this type of ultrasound can also be coadministered
with other therapeutic techniques. However,
most findings indicate that the optimum frequency
and power occurs over a narrow range. We believe that this has been one of the major restrictions to the effective use of low-level therapeutic ultrasound. As a result we
conclude that given accurate dosage and careful administration the use of this methodology will become
widespread.
One of the most important events which has contributed
to the recent development of therapeutic ultrasound occurred in 2001 in Chonqing where a
conference was held on the subject. During the conference a new society devoted to the promulgation of the
general area of ultrasound in non-diagnostic medicine was established under the title "International Society for
Therapeutic Ultrasound". Since then the society has organized other international meetings throughout the world.
Acknowledgement
This work was supported with Natural Science Foundation of China (30200060).
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lntegr Cancer Ther. 2009 Sep;8(3):283-7.
Sonodynamic and photodynamic therapy in
advanced breast carcinoma: a report of 3
cases.
Wang X, Zhang W, Xu Z, Luo Y, Mitchell D, Moss RW.
Chemistry and Environment College, Southern China Normal University, Guangzhou, People's Republic of China. wangxhgz@l63.com
Abstract
Photodynamic therapy (PDT) is an established therapeutic method, first approved by the FDA for certain kinds of cancer in 1998. There are also increasing data to show that a related procedure, sonodynamic therapy (SDT), is a promising new modality for cancer treatment. Here, the authors report clinical results in 3 advanced refractory breast cancer patients who were treated using a combination of sonodynamic and photodynamic therapy (SPDT), along with conventional therapies. All 3 patients had pathologically proven metastatic breast carcinoma. These widely dis­seminated carcinomas had ultimately failed to respond to conventional therapy. A new sensitizing agent, Sonoflora 1 (SF1) was administered sublingually; then, after a 24-hour delay, patients were treated with a combination of light and ultrasound. All patients had significant partial or complete responses. SPDT is a promising new therapeutic com­bination for the treatment of breast cancer.

PMID: 19815599 [PubMed - indexed for MEDLINE]

Mechanisms of Sono Dynamic Therapy
Prog Biophys Mol Biol. 2007 Jan-Apr;93(1-3):212-55. Epub 2006 Aug 8.
Ultrasound-biophysics mechanisms.
O'Brien WD Jr.
Bioacoustics Research Laboratory, Department of Electrical and Computer Engineering, University of Illinois, 405 N. Mathews, Urbana, IL 61801, USA. wdo@uiuc.edu
Prog Biophys Mol Biol. 2007 Jan-Apr;93(1-3):280-94. Abstract
Ultrasonic biophysics is the study of mechanisms responsible for how ultra­sound and biological materials interact. Ultrasound-induced bioeffect or risk studies focus on issues related to the effects of ultrasound on biological ma­terials. On the other hand, when biological materials affect the ultrasonic wave, this can be viewed as the basis for diagnostic ultrasound. Thus, an understanding of the interaction of ultrasound with tissue provides the scien­tific basis for image production and risk assessment. Relative to the bioeffect or risk studies, that is, the biophysical mechanisms by which ultrasound af­fects biological materials, ultrasound-induced bioeffects are generally sepa­rated into thermal and non-thermal mechanisms. Ultrasonic dosimetry is concerned with the quantitative determination of ultrasonic energy interac­tion with biological materials.
Whenever ultrasonic energy is propagated into an attenuating material such as tissue, the amplitude of the wave decreases with distance. This attenua­tion is due to either absorption or scattering. Absorption is a mechanism that represents that portion of ultrasonic wave that is converted into heat, and scattering can be thought of as that portion of the wave, which changes di­rection. Because the medium can absorb energy to produce heat, a tem­perature rise may occur as long as the rate of heat production is greater than the rate of heat removal. Current interest with thermally mediated ultrasound -induced bioeffects has focused on the thermal isoeffect concept. The non- thermal mechanism that has received the most attention is acoustically gen­erated cavitation wherein ultrasonic energy by cavitation bubbles is concen­trated. Acoustic cavitation, in a broad sense, refers to ultrasonically induced bubble activity occurring in a biological material that contains pre-existing gaseous inclusions. Cavitation-related mechanisms include radiation force, microstreaming, shock waves, free radicals, microjets and strain. It is more challenging to deduce the causes of mechanical effects in tissues that do not contain gas bodies. These ultrasonic biophysics mechanisms will be dis­cussed in the context of diagnostic ultrasound exposure risk concerns.

Med Hypotheses. 2009 Apr;72(4):418-20. Epub 2009 Jan 6.
Combination sonodynamic therapy with im‑
munoadjuvant may be a promising new mo‑
dality for cancer treatment.
Ma X, Pan 11, Yi J.
Department of Breast and Thyroid Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Abstract
Sonodynamic therapy (SDT) is a new cancer therapy basing on photodynamic therapy (PDT). Some chemicals pro­duce free radicals on irradiation with laser (photosensitizers) or ultrasound (sonosensitizers). These active molecules destroy biological tissues, thus producing therapeutic effects. Although PDT has been adopted in clinical cancer ther­apy especially for superficial cancers, this modality is under continued investigation for improved efficacy and ex­panded use. For example, PDT-generated tumor cell lysates are effective cancer vaccines; treatment of PDT in con­junction with immunoadjuvant, called "PDT-immunoadjuvant therapy" (PIT), "photoimmunotherapy" or "laser im­munotherapy", is considered to be a promising therapeutic interventions for the treatment of cancers. Ultrasound, especially focused ultrasound, can penetrate deeply into tissues and can be focused into a small region of a tumor to activate the cytotoxicity of sonosensitizers. This is a unique advantage in the non-invasive treatment of nonsuperfi­cial tumors when compared to laser light used for PDT. For the similar mechanism of PDT and SDT, we hypothesize that SDT may be exploited for the generation of effective therapeutic cancer vaccines like PDT; and combination SDT with Immunoadjuvant may be a promising systemic treatment modality, not only for superficial cancers but also for deep-seated tumors, which would surpass PIT.
PMID: 19128891 [PubMed - indexed for MEDLINE]

Ultrasonics. 2008 Aug;48(4):253-9. Epub 2008 Mar 7.
Sonodynamic therapy.
Tachibana K, Feril LB Jr, Ikeda-Dantsuji Y.
Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan, Fukuoka 814-0180, Ja­pan. k-tachi@cis.fukuoka-u.ac.jp
Abstract
Recently, there have been numerous reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles and nanobubbles as carriers/ enhancers of drugs has added a whole new dimension to therapeutic ultrasound. Non-thermal mechanisms for effects seen include various forms of energy due to cavitation, acoustic streaming, micro jets and radiation force which in­creases possibilities for targeting tissue with drugs, enhancing drug effectiveness or even chemically activating cer­tain materials. Examples such as enhancement of thrombolytic agents by ultrasound have proven to be beneficial for acute stroke patients and peripheral arterial occlusions. Non-invasive low intensity focused ultrasound in conjunction with anti-cancer drugs may help to reduce tumor size and lessen recurrence while reducing severe drug side effects. Chemical activation of drugs by ultrasound energy for treatment of atherosclerosis and tumors is another new field recently termed as "Sonodynamic therapy". Lastly, advances in molecular imaging have aroused great expectations in applying ultrasound for both diagnosis and therapy simultaneously. Microbubbles or nanobubbles targeted at the molecular level will allow medical doctors to make a final diagnosis of a disease using ultrasound imaging and then immediately proceed to a therapeutic ultrasound treatment.
PMID: 18433819 [PubMed - indexed for MEDLINE]

Ultrason Sonochem. 2004 Apr;11(2):95-103.
A review of research into the uses of low level
ultrasound in cancer therapy.
Yu T, Wang L, Mason TJ.
Institute of Ultrasound Engineering in Medicine, Chongqing Medical University, Chongqing 400016, PR China. yut­inghe@hotmail.com
Abstract
The use of low power ultrasound in therapeutic medicine is a developing field and this review will concentrate on the applications of this technology in cancer therapy. The effects of low power ultrasound have been evaluated in terms of the biological changes induced in the structure and function of tissue. The main fields of study have been in sono­dynamic therapy, improving chemotherapy, gene therapy and apoptosis therapy. The range of ultrasonic power levels that can be effectively employed in therapy appears to be narrow and this may have hindered past research in the applications in cancer treatment.
PMID: 15030786 [PubMed - indexed for MEDLINEJ