Indiba Summary of the Published and Scientific Evidence...Hepatocarcinoma Cellular Viability (Liver Cancer cells – hepatocarcinoma – HepG2) The aim of this study was to investigate

Indiba : Literature + Scientific Analysis Prof Tim Watson 1

Indiba Summary of the Published and Scientific Evidence

Professor Tim Watson

University of Hertfordshire, UK

June 2017

1 Introduction + Scope Indiba technology covers a wide range of applications from Cell level work in Oncology through the Aesthetics realm applications to Clinical Trials (primarily, but not exclusively in Musculoskeletal applications). This paper aims to provide an overview / summary of the positions in the Cellular and Clinical/Musculoskeletal key application areas. The research in Aesthetics/Beauty therapy is largely omitted from this summary. It is not intended as a full analysis of all the literature, conference presentations, case studies and thesis reports under each heading, but will provide an evidenced and objective overview of the current position from the perspective of an independent academic/researcher.

The Author:

My name is Tim Watson and am currently employed as Professor of Physiotherapy at the University of Hertfordshire, employed on a fractional basis. I have been employed at the University since 1998 and have held my (full) Professorial position since 2003. In addition to my academic research position, I work as a consultant in electrotherapy / electrophysical agents, working for various professional bodies, legal and insurance companies, manufacturers and retail groups undertaking work on a contract basis.

I provide evidence based education programmes in the UK, Europe and Worldwide (over 860 events to date). I have edited a mainstream textbook on electrotherapy (Watson, T. (2008). Electrotherapy: Evidence Based Practice. Elsevier), contributed to several other textbooks and have published over 65 scientific papers of various aspects of electrotherapy and related issues over the last 20+ years. I am currently President of ISEAPT (a formal sub group of WCPT, the International body representing Physiotherapists / Physical Therapists worldwide). I also hold the position of Research Officer for the UK Electrophysical Agents group (EPADU).

I have undertaken a programme of lab based and clinical trial research utilising the Indiba Active device (Section 3 below).

I undertook an analysis of the available literature as part of the FDA submission and CER in 2016 (summarised herein, Section 5).

The views expressed in this paper are my own and reflect an independent stance based on the available evidence rather than from a commercially oriented perspective


2 Cell Research : Dr Alejandro Ubedo + Team, Madrid

The team in Madrid is led by Dr Alejandro Úbeda (at the Ramón y Cajal Health Research Institute (IRYCIS), University Hospital Ramón y Cajal, Madrid, Spain). The researchers at the Institute have developed a range of robust, cell based in vitro experimental models which have evaluated various aspects of Indiba RF safety, cell differentiation, stem cell and repair effects summarised below. The papers associated with this research have been published in peer reviewed, internationally significant journals. The key papers are included in the reference list (Section 6).

In addition to the cytostatic, cytotoxic and cyto differentiation studies, models of the energy transduction pathways have been developed and evidenced such that the primary pathways though which the delivered energy is capable of inducing cell / sub cellular level effects has been identified. This is core work which underpins the gross physiological and clinical trial work reported later in this document.

So far as I am aware, and indeed, Dr Ubeda, this is the only commercially specific RF evidence in relation to cytotoxicity repair and differentiation. Other companies may have conducted equivalent work, but it does not appear in the published / public domain.

2.1 Cell Studies in Cytostasis / Cytotoxicity

[Def: Cytostasis : is the inhibition of cell growth and multiplication. Cytostatic refers to a cellular component or medicine that inhibits cell growth. Cytostasis is an important prerequisite for structured multicellular organisms : tending to retard cellular activity and multiplication]

[Def: Cytotoxicity: the quality of being toxic to cells]

[Def: Oncostatic : that halts the spread of a cancer]

The use of capacitive resistive energy (Indiba equivalent) in the 0.45 – 0.60MHz range has been shown the have an apparently inhibitory effect on the rate of growth of tumours. The early work, in the 1990’s was conducted by Ley et al (1992, 1993, 2003; published in Spanish) and subsequently by researchers in Japan (Sakamoto et al (1997); Matsui et al (2000), Ohguri et al (2004)). The group in Madrid aimed to investigate this effect and further, establish the cellular and molecular mechanisms which might lead to these apparent cytostatic/cytotoxic effects.

Important work involved the in vitro investigation of Indiba RF application(s) and its effect on

a) Cell death and viability b) Cell proliferation and differentiation c) Transduction pathways

If the application of the RF energy led to cell death, this would constitute a detrimental effect (in terms of a therapy application – though it has strong potential in an oncology environment – usefully considered in Hernandez-Bule et al, 2007).


Cell Study Model

A cellular model was established in which the RF energy was passed through cells in a Petri dish containing a cell culture. This basic model has been employed by the research group in Madrid (with slight variations) in several published research reports.

The first stage was to identify the ‘dose’ (current density) which could be passed through the culture without significant thermal effect. This was identified as being between 1 – 100 μA/mm2 , 50μA/mm2 being employed in subsequent experimentation (Figure 2.1).

Figure 2.1: Subthermal current density for in vitro experimental model (from Hernandez Bule et al, 2007)

The developed protocol involved 1 x 5 minute RF energy exposure every 4 hours over a period ranging from 12 – 48 hours. The subsequent measured cellular effects would not be generated by a thermal response (which is well established in cell biology) – and thus were attributable to ‘non-thermal’ RF mechanisms.

Hepatocarcinoma

Cellular Viability (Liver Cancer cells – hepatocarcinoma – HepG2)

The aim of this study was to investigate the response from human hepatocarcinoma HepG2 line, during and after exposure to Indiba equivalent CRET currents (0.57MHz) at subthermal densities. It was hypothesised that the RF energy application at subthermal levels would result in decreased proliferation and potential cytostasis of the hepatocarcinoma (HepG2) cells.

The method (summary) involved a comparison of exposed vs sham exposed cell cultures. Both cell groups were grown for a 4 day period under controlled conditions. Commencing on Day 4, cells were exposed (or sham exposed) to the energy. Treatment was applied for 5 minutes every 4 hours over a 24 hour period. Methodologically, several confounding variables were managed including artefact control; influence of the electrodes; electrode electrochemistry; electromagnetic field effects; electrophoresis effects; thermal effects. Details in the full paper.


Figure 2.2: Basic set up (from Hernandez Bule et al, 2007)

The results (2007) demonstrated that exposure to the RF currents induced a decrease cell population and protein content, with maximal effect some 24 hours after the exposure. The intervention did not induce cell death but did appear to have a blocking effect on cell proliferation.

A further paper (Hernandez-Bule et al 2010) evaluated the cytostatic response of hepatocarcinoma (HepG2) cells to 0.57

(Indiba equivalent) currents when compared with a sham exposure. The basic in vitro model was as described above. The study aimed to evaluate the anti proliferative effect of the intervention on HepG2 cells. The results (summary) demonstrated a significant change in the expression and activation of cell cycle control proteins (D1, A, B1), and that this change was responsible for the demonstrated antiproliferative effect. The paper further contributes to the transduction model (section 2.2.2 below).

In Summary : (2007 and 2010 papers)

• Short applications CRET (Indiba equivalent) current at subthermal densities exert a anti-proliferative effect in human cancer cells

• Such effect is due to slowdown of the cell cycle (phases G1 and S) • Slowdown is due to electrically-induced changes in the expression of proteins involved in cell

cycle regulation (D1, A, B1) • Being the cell membrane protein C-met a potential primary target and physico-chemical

transducer of the electric stimulus

Neuroblastoma

Hernandez-Bule et al (2012) carried out some work employing 0.57MHz currents and evaluating the response of neuroblastoma cells (NB69)as a follow up to their positive preliminary study in neuroblastoma study (Hernandez-Bule et al (2004).

The 2012 study demonstrated that the applied currents at subthermal levels has a cytotoxic effect on the neuroblastoma cells. The mechanism of action involved the cell proliferation capability of this cell line, with a reduction of the fraction of cells in the G2/M phase at 12 hours of exposure.

The authors suggest that this response can be considered to be cytotoxic and/or cytostatic.

Importantly, in the same study, human blood mononuclear cells were exposed to the same energy regime, but demonstrated no response. This is a positive finding – in that the applied energy has the capacity to influence (downregulate) the proliferation of cancer (neuroblastoma) cells, whilst having no measurable effect on ‘healthy’ (blood) cells.


In Summary :

• Subthermal application of Indiba equivalent currents has a cytotoxic effect on neuroblastoma cells

• The applied energy had an inhibitory effect on (neuroblastoma) cell proliferation • The applied energy did not have an inhibitory effect on healthy (blood) cells

2.2 Physical – Chemical Transduction

Linked with the previous studies, and backed up by additional work, the transduction pathways for these effects has been proposed. Essentially, this involves identifying the mechanism by which the application of a stimulus (in this case the Indiba energy) can bring about the changes in cell function which have been observed. The primary reference for this published work is in Hernandez-Bule et al (2014 a) with additional material in Hernandez-Bule 2007, 2010.

So far as I am aware from my search of the literature, this pathway HAS been established for the Indiba energy application and not for competitor devices.

A summary of the proposed pathway(s) is shown in Figure 2.3.

Figure 2.3: Proposed transduction pathway for Indiba in terms of cell proliferation and apoptosis effects.


In Summary:

The results were not indicative of cancer damage due to the INDIBA electric current,

The results were demonstrative of an oncostatic action of the electrical (subthermal) INDIBA signal

2.3 Cytodifferentiation

[Def : (1) a process by which embryonic cells acquire biochemical and morphological properties essential for specialization and diversification. (2) the total and gradual transformation from an undifferentiated to a fully differentiated state.]

Stem cells are single undifferentiated cells which can replicate and can (importantly) differentiate into a wide variety of specialist cell types – such as muscle, bone, cartilage etc. They are often referred to in this regard as pluripotential.

In current medical practice, the potential for using stem cells as a therapeutic tool has gathered significant momentum in recent years, with obvious and very attractive treatment potential. Many research groups in numerous medical specialities are investigating how this type of application might be put to best use.

In order for a stem cell to differentiate into a particular type of specialist cell, a stimulus is required. It is possible that the electric currents delivered through the Indiba system could act as such a stimulus.

2.4 Adipocyte Differentiation, Fat Deposition and Lipolysis

Adipocytes are specialist cells which store fat inside cytoplasmic vesicles.

It is possible (based on the work of Kato et al in Japan), that Indiba energy delivered at high dose (thermal level) is sufficiently strong as to induce lipolysis [Lipolysis is the breakdown of lipids into glycerol and free fatty acids]. This being the case, it has clear and advantageous potential in aesthetic applications which are largely outwith the remit of this summary paper.

It was proposed that using the same (Indiba) energy at lower (non thermal) doses may also bring about effects on adipocytes, and Hernandez Bule et al (2016) report on such an intervention

Stem cells were incubated for a period of either 2 or 9 days. During the last 48 hours of incubation, samples were exposed to verum or sham Indiba at low (non thermal) current density for a period of 5 minutes every 4 hours (as per the previous protocol).

The results demonstrated that the application of Indiba reduced lipid deposition in the early and intermediate stage of stem cell differentiation – effectively this is an anti-adipocytic effect – which clearly has strong potential in aesthetics.

The team went on to establish the mechanism by which the application of Indiba current achieved this effect. Put simply, the treatment effectively brought about a significant sub-expression of 4 genes which influence the storage of fat (PPARG1; PLIN; ANGPTL4; FASN).


[a more complete explanation]

During adipogenesis, P PAR gamma is present at the nuclear level. At the nucleus, PPARγ controls the TRANSCRIPTION of the RNA that encodes for relevant adipogenic proteins, like FASN, Angiopoietin 4 or Perilipin. These proteins exert crucial roles to the synthesis and preservation of the adipose tissue. For example, Perilipin preserves the fat from degradation by lipolytic enzymes present in the cells. Thus, a blockade at any step of the process that leads to the synthesis of this protein would expose the fat to the lipolytic enzymes and, thus, would have an anti-adipogenic effect.

On exposure to Indiba currents there is an increased expression and activation of P MEK one and two at the cytoplasm of the cell. Then, P MEK enters the nucleus and expulses P PAR gamma to the cytoplasm. The P PAR gamma disappears from the nuclei and is moved to the cytoplasm where it degrades. This constitutes an antiadipogenic effect.

In Summary :

• At the early and intermediate adipogenic phases, the Indiba signal impairs / inhibits the formation and preservation of lipid vesicles (i.e. limits fat storage capability).

• In mature adipose cells the electric stimulus could induce fat degradation or mobilization. • Additionally, at higher doses (not these investigations) the effects identified above could act

to sensitize (increase vulnerability of) the adipocytes to the lipolytic actions of the treatment. The thermal and mechanical effects of Indiba treatment bring about lipolysis (

• Given that the Indiba applied current penetrates deep into the tissue, it could have an effect on both the subcutaneous and deep (visceral) fat.

2.5 Stem Cell Proliferation for Tissue Regeneration

Tissue regeneration (as opposed to tissue repair) is a phenomena which has been investigated over the last 5 or 6 decades. It offers the potential to replace damaged tissue rather than ‘mend’ it with scar tissue. This is an inherently attractive proposition in that a regenerated tissue would have characteristics more akin to the original tissue prior to damage or disease. Scar tissue can bring about an effective structural repair, but the characteristics (and thus, function) of scar tissue is not the same as the muscle, ligament or cartilage that is replaces.

There is a strong body of evidence that demonstrates a significant relationship between tissue regeneration and electrical stimulation of various types. This concept and the evidence behind it has been reviewed numerous times, including Watson (208) and Kloth (2005.)

Dr Ubeda and the Madrid team have investigated the potential for Indiba based therapy to influence this tissue regeneration process.

After obtaining informed consent from male and female patients undergoing minor surgery, small pieces of disposable tissue containing a variety of cell types, including a few stem cells, were collected. The tissue was processed, digested and centrifuged in the lab in order to select and isolate the stem cells which were seeded in Petri dishes.


The stem cells were incubated for periods of 2 or 9 days. During the last 48 hours of incubation the samples were exposed, or sham-exposed, to 5-minutes of INDIBA at non-thermal intensities. Each 5 minute exposure was followed by a 4-hour interval of no exposure as per previous experimental protocols.

Results:

Exposure to the Indiba current(s) resulted in an increase in the number of cells in the proliferative phases (S phase: 120.6 ± 8.3%; G2/M phase: 109.9 ± 2.9%) which is a statistically significant % increase over the control (sham) exposure.

The mechanism by which this effect is achieved is demonstrated to be the ERK MAP Kinase (MAPK-ERK1/2) pathway.

Full details of this experimental work are found in Hernandez Bule et al (2014 b)

In summary, this experimental work demonstrates that the Indiba treatment has the capability of increasing the proliferative potential of an exposed tissue (based in in vitro work on cell cultures).

2.6 Extending the Stem Cell Model Work : Cartilage, Bone, Skin

Work in the Madrid team includes an investigation of chondrocyte differentiation. Chondrocytes are cells which secrete the matrix of cartilage and become embedded in it.

It is possible that the application of Indiba currents could facilitate the differentiation of stem cells into chondrocytes and/or could promote the synthesis (generation) of new cartilage by chondrocytes.

Clearly this has a range of significant medical applications ranging from osteoarthritis management through to acute cartilage damage following injury.

The early results indicate that Indiba based currents serve to stimulate the (molecular) processes which lead to chondrocyte differentiation. The level of this effect has been shown to be at the protein synthesis level, not in terms of gene expression.

This alone is potentially distinctly advantageous.

There is a second possibility (as above) in that the Indiba treatment may serve to enhance the secretion of the extra cellular matrix (ECM) by the chondrocytes (whether those already in the tissue or those newly differentiated as a result of the process described above).

The preliminary results indicate that the application of Indiba promotes the synthesis of GAG’s (glycosaminoglycans) which are integral to cartilage structure and function. The study continues and now the primary target is to evaluate whether the Indiba treatment results in enhanced collagen synthesis.

In summary : IF the Indiba intervention is capable of promoting stem cell differentiation into chondrocytes and stimulating chondrocytes (new or existing) to produce additional GAG’s and collagen, the potential for future clinical use is very substantial with the capacity to be of benefit to a large number of patients with degenerative joint pathologies and those with more acute cartilaginous injuries.


Clearly, this avenue of research will be extended to other tissues – most obviously BONE (stimulating osteocytes and thereby osteogenesis) and SKIN (stimulating keratinocytes) both in terms of their proliferation and also their migration into the regenerating tissue.

2.7 Summary of the Dr Ubeda Madrid Team Research

Cancer cells and potential cell damage:

• Short exposures to Indiba current at subthermal densities exert an anti-proliferative effect in human cancer cells (hepatocarcinoma, neuroblastoma).

• Such effect is due to slowdown of the cell cycle (phases G1 and S). • Slowdown is due to electrically-induced changes in the expression of proteins involved in cell

cycle regulation. • The cell membrane protein C-met is a potential primary target and physico-chemical

transducer of the electric stimulus. • The same energy applied to healthy cells (human blood) has no inhibitory effect on cell

behaviour.

Adipocyte Differentiation and Antiadipositic Effect

• At the early and intermediate adipogenic phases, the Indiba signal impairs / inhibits the formation and preservation of lipid vesicles (i.e. limits fat storage capability).

• In mature adipose cells the electric stimulus could induce fat degradation or mobilization. • Additionally, at higher doses these effects could act to sensitize (increase the vulnerability

of) the adipocytes to the lipolytic actions of the treatment. The thermal and mechanical effects of Indiba treatment bring about lipolysis.

• Given that the Indiba applied current penetrates deep into the tissue, it could have an effect on both the subcutaneous and deep (visceral) fat.

Stem Cell Proliferation and Tissue Regeneration

• This experimental work demonstrates that the Indiba treatment has the capability of increasing the proliferative potential of an exposed tissue.

• Exposure to the Indiba current(s) resulted in an increase in the number of cells in the proliferative phases at a statistically significant % increase over the control (sham) exposure through the MAPK-ERK1/2) pathway.

Stem Cells and Chondrogenesis

• Indiba currents serve to stimulate the (molecular) processes which lead to chondrocyte differentiation. The level of this effect has been shown to be at the protein synthesis level.

• Additionally, the data indicate that the application of Indiba currents promotes the synthesis of GAG’s (glycosaminoglycans) which are integral to cartilage structure and function.

• The possibility that treatment with Indiba currents therefore enhances stem cell differentiation into chondrocytes, and also stimulates chondrocytes (new and existing) to synthesize GAG’s and collagen has strong and significant clinical potential.

Stem Cells, Bone and Skin


• Preliminary work supports the hypothesis that along the same lines as in cartilage (with chondrocytes), Indiba currents have the potential to enhance bone development (by means of an osteocyte mechanism) and skin repair (by means of a keratinocyte mechanism). Both have highly significant clinical potential value.

The work is published in highly regarded, peer reviewed journals.

3 Prof Tim Watson+ Team, University of Hertfordshire, UK

The research group at the University of Hertfordshire have two research foci of relevance in the context of Indiba treatment applications: (1) Lab based research to establish gross physiological effects of a treatment when delivered to healthy individuals and (2) Clinical trial(s) involving the delivery of electrophysical modalities with patients exhibiting a range of clinical presentations.

3.1 Literature Review(s) Two Narrative Reviews have been undertaken and published concerning the use of Radio Frequency (RF) based therapies in current practice. Therapists are familiar with shortwave based RF frequency application – in both continuous and pulsed modes – and these reviews set out to identify the range of literature relating to these applications, but additionally set out to demonstrate that shortwave frequencies (commonly 27.12MHz) were not the only clinically available frequencies, and therefore to raise the awareness of non-shortwave frequency evidence, which clearly includes the Indiba RF applications at approximately 0.5MHz.

The two review papers covers RF applications in Acute (Part I) and Chronic (Part II) conditions.

There is a relatively small volume of literature pertaining to the non-shortwave applications, but what there is in the published domain was integrated into the review material.

These publications ‘set the scene’ for the Indiba related work which followed.

In Summary : The reviews were supportive of RF applications in therapy and offered a rationale and context for Indiba related applications from an objective standpoint whilst identifying the need for further work at these low RF frequencies. [Kumaran and Watson (2015a and 2016)]

3.2 Lab Based Studies at the University of Hertfordshire In order to establish robust evidence for the physiological effects of Indiba RF application(s) in a healthy population, a series of lab based studies were carried out at the University of Hertfordshire, integral to the PhD work of Binoy Kumaran. Not only were these beneficial as a component of the Indiba evidence platform, they were considered essential in order to achieve Ethical clearance for the clinical trial which was the final element of Kumaran’s PhD related studies.

The essential components of the lab studies are outlined below. Several papers have been published in peer reviewed journals and presentations at (peer reviewed) National and International conferences as a mean to disseminate the outcomes of this work. The studies are strongly supportive of real and significant effects of Indiba based therapy physiological effects. The


comparison with the nearest employed therapy (shortwave in pulsed mode) identify that the Indiba effects are (a) more pronounced and (b) longer lasting than those currently available.

3.2.1 Energy Levels applied and Associated Thermal Effects

The first main lab study was to evaluate the thermal onset levels with Indiba applied in CAP and RES modes on a healthy population employing an RCT design, adequately powered to demonstrate any significant changes.

The main aims of this study were to:

1) To identify the point of onset of heating, the point of optimum heating, and the point of onset of heat discomfort with the application of either mode of CRMRF (Indiba) therapy.

2) To record the baseline skin temperature, to record the immediate post treatment skin temperature and to periodically record the subsequent post treatment decline in skin temperature (thermal decay process) at the treated area

A sample of 15 participants were recruited for this study.

All participants were exposed to a CAP and a RES energy exposure on different occasions, separated by an interval sufficient to allow wash out (based on pilot experimentation).

Essential procedure (full details available): The return electrode was positioned under the calf muscle belly. The Indiba treatment was applied within a marked area on the anterior lower thigh (just above the knee) using the active electrode and 20 ml of conductive cream. For either mode of treatment (CAP or RES), the intensity of delivery started at the minimum permitted level on the device and was raised by one level every 30 seconds. The active electrodes was moved in a circular pattern as would be the case with a clinical application.

During treatment the participants reported clearly and promptly at three time points: Firstly, at the onset of heating of the skin (thermal onset), secondly, when the heat builds up to a moderate (yet comfortable) level (definite thermal sensation), and thirdly, at the point when the heat starts to cause discomfort (onset of thermal discomfort). The three time points and the corresponding Indiba intensity were noted. The treatment was promptly stopped once the ‘onset of thermal discomfort’ was reached. The temperature was recorded from the treated and the untreated limb. Core temperature was additionally monitored.

Both modes of the Indiba treatment were well tolerated, with no accounts of any undesirable incidents that might have resulted directly or indirectly from the treatment, including potential concerns due to overheating or any other delayed tissue reactions. All 15 participants completed both their treatment sessions and assessments.

The summary results indicate that skin temperature increased in both CAP and RES mode. The tissue temperature increase was more pronounced in RES mode. The temperature changes were still significantly greater than baseline 45 minutes after cessation of the therapy. (Figures 3.1 and 3.2). There was no change under control conditions (the untreated limb) and there was no change in core temperature.


Figure 3.1: Summary results for skin temperature changes following Indiba therapy delivered in CAP and RES modes

Figure 3.2: Illustration of thermal decay profile for Indiba therapy delivered in CAP (green) and RES (blue) modes

In summary : the study demonstrated that the skin temperature is significantly increased in both CAP and RES modes, though the RES mode changes were greater. There was a significant difference between the thermal response patterns in CAP and RES modes, with the RES mode changes being both greater and sustained for a longer period post intervention.

The results have been published in the International Journal of Hyperthermia (2015) (Kumaran and Watson 2015 b)


3.2.2 The Effect of Indiba Active on Key Physiological Outcomes in a Healthy Population

The key concept in this experimentation was to measure Indiba treatment related physiological effects (skin temperature, skin blood flow, deep tissue blood flow, nerve conduction velocity and tissue extensibility, core temperature, blood pressure) and to compare the effects with those achieved with pulsed shortwave therapy at the nearest equivalent dose.

The procedures were similar to those reported above, with the addition that an ultrasonography device (Esaote MyLab70 XVG ) was employed to measure the deep blood flow and tissue extensibility. The Biopac functions were extended to record nerve conduction velocity.

Formally, the aims were :

1) To compare the effects of multiple doses of Indiba CRMRF therapy on SKT, skin and deep blood flow, NCV and extensibility of tissues on a group of asymptomatic adults in a randomised crossover study.

2) To compare the above effects with those obtained from a high dose PSWT treatment in the same group of people.

A sample of 18 participants were recruited for this study which (using a power calculation) was adequately powered to detect significant effects if they were present.

Treatment was delivered on 5 separate occasions constituting: (1) Indiba High Dose (evident thermal perception), (2) Indiba Low Dose (sub/minimally thermal). (3) Indiba Placebo (no energy delivered), (4) Control condition and (5) High dose Pulsed Shortwave.

Figure 3.3: Experimental procedure for physiological evaluation of Indiba and Pulsed Shortwave interventions

The results are summarised in the Figures below:


Figure 3.4: Skin Temperature changes with Indiba and Pulsed Shortwave treatments

Figure 3.5: Skin Blood Flow changes with Indiba and Pulsed Shortwave treatments

Figure 3.6: Deep Tissue Blood Flow changes with Indiba and Pulsed Shortwave treatments


The deep blood flow velocity – no significant change under any treatment condition – therefore blood flow VOLUME and INTENSITY significantly increased, but the flow rate (velocity) was not significantly altered.

Nerve Conduction Velocity : no significant change under any treatment condition

Tissue Extensibility: No significant change between conditions

Blood Pressure, Core Temperature: No significant change under any tested condition

In Summary : The results of this study suggest that a high (thermal) dose as well as low (sub/minimally thermal) dose of Indiba can significantly enhance and sustain local Skin Temperature, while only the high dose has a meaningful impact on Skin Blood Flow. An equivalent high dose of Pulsed Shortwave increased the Skin Temperature marginally, but did not sustain it over the follow-up phase. Pulsed Shortwave also failed to show any meaningful effect on the Skin Blood Flow.

The results also suggest that the high and low dose treatments of Indiba can significantly enhance blood flow volume at depth, while only the high dose can significantly enhance both volume and intensity of flow. An equivalent high dose of PSWT failed to show any impact on any of the deep blood flow measures.

None of the treatment groups had a statistically significant impact on the velocity of deep blood flow, although the high dose RF increased the velocity marginally. The hardness and softness of tissues, Nerve Conduction Velocity, Heart Rate and Blood Pressure were also not influenced by a local therapeutic application of either type of RF therapy. The untreated contralateral leg did not show any meaningful change in its physiological responses.

These results not only demonstrate significant and clinically relevant tissue changes as a result of a 15 minute Indiba treatment, but also demonstrate a clear advantage of the Indiba treatment over an equivalent pulsed shortwave intervention (the nearest currently employed clinical equivalent).

The results of this study have been published in the European Journal of Physiotherapy (Kumaran et al 2017) and also reported at the World Physical Therapy Congress (Kumaran and Watson 2015 c) and the UK Physiotherapy conference (Kumaran and Watson 2015 d)

3.3 Clinical Trial – Osteoarthritis of the Knee A final study as part of the Kumaran PhD research was a clinical trial involving patients with Osteoarthritis of the Knee – by way of a typical chronic clinical group who might benefit from this thermal based intervention.

The trial was carried out at a UK NHS hospital (therefore representing a ‘true’ group of these patients) and was sufficiently powered to detect significant clinical treatment effects if they were present. Forty two patients were recruited in this trial.

The trial is reported in full in the PhD thesis and has been submitted to the Journal of Physiotherapy where it is currently under review.

There were 3 groups in the clinical trial (a) High dose Indiba + usual care (2) Placebo Indiba + usual care and (3) Usual care only. The aim of this design was to be able to evaluate the added benefit of


Indiba based therapy over and above usual care and to be able to identify a real treatment effect over and above the placebo response. Patients in the Indiba High and the Indiba Placebo groups received treatment (15 minutes) 2 x weekly for 4 weeks (reflecting a ‘normal’ clinical protocol).

Formally, the aims were to investigate:

1) Whether 448 kHz Indiba treatment can reduce symptoms and significantly improve function in patients affected by chronic OA of the knee joint(s).

2) Whether any such potential clinical benefit is significantly better than a placebo treatment or the current standard treatment for OA that is based on exercise and advice.

The physiological responses demonstrated in the patient group were measured on one occasion (the first visit) so that a comparison could be made between healthy participant and patient responses to the same treatment. This aspect of the work is reported (briefly) here and will be submitted to an appropriate journal at some point in the near future.

Figure 3.7: Basic design of the Indiba OA Knee clinical trial

In addition to the physiological parameters employed in the lab based research, a range of clinically appropriate outcomes were employed including pain, function, quality of life and range of movement – these being representative of the normal clinical outcomes used for this patient group in other equivalent research. It is intended to make a comparative analysis of the Indiba outcomes with those from other trial involving (for example) ultrasound, TENS, NMES etc. This work is currently underway.

Pain


Figure 3.8: Pain responses demonstrating a clear (significant0 difference between the Indiba High and other treatment groups.

The reduction in pain experienced by patients in the Indiba group was significantly greater than for those in the placebo or control (current care) groups. The pain improvements were significant at statistical and clinically meaningful (MCID) levels.

WOMAC (Function and Quality of Life)

Figure 3.9: WOMAC scores for the clinical groups demonstrating a significant advantage to those in the Indiba High group

The change (improvement) in WOMAC score for patients in the Indiba group was significantly greater than achieved in the placebo or control (current care) groups. The improvement was at a clinically meaningful level.


In Summary : A four-week high dose Indiba intervention (that produced a moderately thermal response) delivered alongside exercises and advice about self-management of OA knee resulted in a statistically significant and clinically meaningful reduction of pain as well as improvement of function in patients affected by chronic OA in their knee joints.

The effects were significantly greater compared to those obtained using the current standard care comprising exercises and advice.

The treatment effects were significantly maintained at 1 and 3 month follow up time points.

A placebo intervention when delivered in addition to standard care, also induced significant clinical benefits (as expected) when compared to the standard care alone; however, the size of the effect was significantly smaller than that obtained with the active Indiba treatment.

4 Other Clinical Application Data

Indiba has accumulated a range of literature relating to the use of Indiba technology (and it’s predecessors). Some of this material has been presented at conferences, some has been provided to the company by way of a report, some is published in journals and some pertains to a thesis (various levels from BSc through MSc to PhD). This material covers a wide range and is summarised here rather than a paper by paper analysis (over 150 items fall under this heading, 172 of which were considered for the CER analysis (Section 5).

Taking this literature in clusters, the broad categories and the dominant content of each are highlighted below

General Reviews and Reports (56)

In this group are numerous papers which consider the use of Indiba based therapy across a clinic, clinical setting or client group. So for example, there might be a report on the use of Indiba in acute and chronic sports injuries.

This group covers a wide range of applications. They are almost exclusively supportive of the therapy. The majority have not been published in journals though numerous have been presented at conferences.

Musculoskeletal and Orthopaedic (27)

This group, excluding those specifically related to soft tissue/sport related injury is dominated by back pain, neck pain and osteoarthritis (totalling 21/27). They are supportive of Indiba based therapies.

Sport Related and Soft Tissue Injury (24)

In addition to several general papers (included in the first section), there are a good range of papers, reports and conference (poster) presentations which are specifically concerned with patients in this group. The dominant classifications within which are pertaining to muscle and ligament issues (15/24), which is a fair reflection of the dominance of these clinical issues in sport and soft tissue clinics. The studies are strongly supportive of the benefits of Indiba based therapies.


Animal, Cell and Oncology related (24)

Numerous of these are published papers and have been considered in some more detail in the preceding section(s). Work relating one way or another to cancer, malignancy and associated issues dominate with 18/24 papers. Many have been published/presented in a peer reviewed environment, and are supportive of beneficial effects having been demonstrated.

Aesthetics (9)

Much as the aesthetics and beauty arenas have been largely excluded from this summary report, there are some resources within the Indiba library which pertain to the use of Indiba based therapies in this area of practice. Some have been published. Based on my consultation of the literature generally within the electrophysical arena, I would consider this body of work to represent a small proportion of the available material. Those which are included in this group are supportive of beneficial effects.

Animal (Veterinary) Based Therapy (7)

This is a growing body of literature / report / theses, mainly dealing with equine and canine practice in which the reports are supportive of benefit. In some of my current work with the animal therapy based literature, I would anticipate distinct growth in this therapy context. The papers in this group are supportive of Indiba intervention in animal health presentations.

Others

The remaining papers cover a disparate range of application running from spasticity management in children through to lymphoedema and phantom limb pain (plus many more). Whilst many of these topics may only have a single paper/report, they are almost exclusively supportive in their conclusions.

5 Summary of the Literature Analysis for the CER (2016) and FDA Submission

I carried out an analysis of the available literature in 2015/6 under 4 specific indications for the purposes of a CER report prior to a submission to the FDA. The indications were :

• Relief of pain, • Relief of muscle spasms, • Increase in local circulation, • Temporary reduction in the appearance of cellulite

An independent search of the literature was undertaken and the resulting publications were utilised in conjunction with eligible Indiba company reports and associated material. Databases searched included : Medline/Pubmed, ISI Web of Science, Scopus, CINAHL and the Cochrane Database.

From this large body of literature, 51 papers were included in the detailed analysis.

I will not replicate the detailed analysis here, but summarise under the indications identified.


Ache/Pain

28 papers were included in this group (16 journal/conference papers, 7 Indiba internal reports and 5 theses. These included RCT’s, controlled trials, cohort studies and Case Studies/Series. Additionally, material from 4 reviews were considered.

A total of 1475 patients were involved in these research activities, 1204 of whom were in receipt of RF energy applications.

Overall, 22/28 (79%) papers demonstrated statistically significant pain relief and 20/28 (72%) further demonstrated pain relief at a clinically significant level.

There was nothing in the reviewed literature which indicated evidence of significant adverse effects or responses.

Overall, the material in this section was strongly supportive of the use of RF energy as a means to effectively bring about pain relief.

Increase in Local Circulation

4 papers were included in this group (all RCT’s in healthy populations) Additionally, material from 1 review was considered.

A total of 51 participants were involved in the research activities and all 51 were exposed to RF energy.

Overall, 4/4 (100%) papers demonstrated statistically significant increase in local blood flow. It was not possible to determine clinical significance as all trials involved healthy participants.

There was nothing in the reviewed literature which indicated evidence of significant adverse effects or responses.

Overall, there is sufficient evidence to support this intervention as a means to stimulate a local circulatory response (increase local blood flow).

Cellulite

5 papers were included in this group (1 controlled trial and 4 cohort studies) Additionally, material from 9 reviews were considered.

A total of 109 participants were involved in the research activities and all 109 were exposed to RF energy.

Due to methodological constraints, it was not possible to report (or calculate) statistical significance, but it is implied in the reported results.

2 papers report (minor) adverse effects which resolved without any significant intervention.

The balance of research evidence in the included papers is supportive of this indication (to reduce or alter the appearance of cellulite). These findings are consistent with the broader picture presented in the review material


Muscle Spasm

There were no Indiba specific research papers dealing with this indication though it is included in 13 reviews which were analysed.

Whilst no Indiba specific research is presented in direct support of this indication, it is argued from the literature that there is a well established relationship between muscle spasm, heat, pain, circulatory changes and local blood flow. The Indiba family of devices are evidenced as having significant effects on local tissue temperature, blood flow and pain and thus one would anticipate a significant effect on local muscle spasm in line with other thermal based applications.

It was concluded that there is supportive evidence (direct and indirect) that the Indiba family of devices, delivering RF energy in resistive and capacitive modes as described, can achieve a significantly beneficial effect on ache/pain, local circulation, cellulite and the relief of muscle spasm.

6 Overall Summary + Conclusion This report does not claim to have analysed in detail, nor indeed, reported on all the published and unpublished material pertaining to Indiba based therapy. It does represent an objective view of the material of which I am aware, have found or has been provided to me. I have not attempted in any way to exclude publications or reports which have negative outcomes and therefore bias the report contents, though there are few items which provide negative or ‘no difference’ outcomes.

The cell based work, currently centred with Dr Ubeda and his team in Madrid continue to provide strong evidence of positive and beneficial cell and sub cellular level effects. The application of Indiba based therapy has a strong potential to influence the development of cancer cells, effectively slowing their development and likely slowing or stopping the growth and spread of tumour tissue. Whilst this is predominantly lab based, in vitro research, and does not immediately transfer into the clinical environment, I am not aware of equivalent research with other similar devices. The evidence is strong, published in high profile peer reviewed internationally reputable journals.

The Lab and Clinical studies from the Hertfordshire group, which I lead, is also strong and provides robust evidence of a real physiological, dose dependent response to the application of Indiba currents into the tissue. These effects are significantly stronger than those achieved by a placebo application and furthermore, more pronounced than those achieved by the currently available nearest clinical equivalent therapy (pulsed shortwave therapy).

The clinical trial result with OA knee provides evidence of a statistically and clinically significant beneficial effect when the therapy is compared with a placebo intervention and current best evidenced care. The results of the clinical trial do not suggest that Indiba should be employed instead of current therapy, but that its addition to the treatment package adds significant benefit.

The literature review undertaken as part of the CER/FDA submission (summarised in Section 5) is supportive of Indiba based therapies across a range of indications. A volume of literature was consulted and analysed in the preparation of that document which has not been replicated in this summary document.

Finally, a range of material in the ‘Indiba Library’ covers applications across musculoskeletal, sport, aesthetics, animal (veterinary), cell and other application arenas. The vast majority of this material is


supportive of the intervention, and whilst only a proportion has been either published in journals or presented at a relevant conference, it is difficult to ignore such a substantive body of work.

I would conclude that there is a supportive evidence base for the use of Indiba RF currents across a wide range of applications. At some point in the future, with additional and focussed research, some of these areas/applications are likely to become more dominant, and much as I could speculate as to what they might be, I have tried throughout this summary to be objective rather than speculative.

Prof Tim Watson

May 2017


7 References

Hernandez-Bule, M. L., M. A. Cid, M. A. Trillo, J. Leal and A. Ubeda (2010). "Cytostatic response of HepG2 to 0.57 MHz electric currents mediated by changes in cell cycle control proteins." Int J Oncol 37(6): 1399-1405.

Hernández‑Bule, M. L., J. Martínez‑Botas, M. Á. Trillo, C. L. Paíno and A. Úbeda (2016). "Antiadipogenic effects of subthermal electric stimulation at 448 kHz on differentiating human mesenchymal stem cells." Mol Med Rep 13(5): 3895-3903.

Hernández-Bule, M. L., C. L. Paíno, M. Á. Trillo and A. Úbeda (2014 b). "Electric Stimulation at 448 kHz Promotes Proliferation of Human Mesenchymal Stem Cells." Cellular Physiology and Biochemistry 34(5): 1741-1755.

Hernandez-Bule, M. L., E. Roldan, J. Matilla, M. A. Trillo and A. Ubeda (2012). "Radiofrequency currents exert cytotoxic effects in NB69 human neuroblastoma cells but not in peripheral blood mononuclear cells." Int J Oncol.

Hernandez-Bule, M. L., M. A. Trillo, E. Bazan, M. A. Martinez-Pascual, J. Leal and A. Ubeda (2004). "[Nonthermal levels of electric currents applied in capacitive electric transfer therapy provokes partial cytotoxic effects in human neuroblastoma cultures]." Neurocirugia (Astur) 15(4): 366-371

Hernandez-Bule, M. L., M. A. Trillo, M. A. Cid, J. Leal and A. Ubeda (2007). "In vitro exposure to 0.57-MHz electric currents exerts cytostatic effects in HepG2 human hepatocarcinoma cells." Int J Oncol 30(3): 583-592.

Hernandez-Bule, M. L., M. A. Trillo and A. Ubeda (2014 a). "Molecular mechanisms underlying antiproliferative and differentiating responses of hepatocarcinoma cells to subthermal electric stimulation." PLoS ONE 9(1): e84636.

Ley, A., J. M. Cladellas, S. Colet, P. De las Heras, R. Florensa, J. Prim, J. Roussos, A. Ariza and J. Calbet (1992). "Transferencia eléctrica capacitiva (TEC). Técnica no invasiva de hipertermia profunda en el tratamiento de los gliomas cerebrales. Resultados preliminares (Spanish)." Neurocirugía 3(2): 118-123.

Ley, A., A. Ariza and R. Rosell (1993). Tratamiento quirúrgico de los gliomas malignos. Hipertermia (In Spanish). Tumores del Sistema Nervioso Central. Epidemiologia, Nosología y Terapéutica. Doyma. Barcelona: 55-64.

Ley-Valle, A. (2003). "Hipertermia intracraneal no invasiva mediante la técnica de Transferencia Eléctrica Capacitiva-TEC-(*). Resultados de la termometría cerebral e intratumoral." Neurocirugía 14(1): 41-45.

Kloth, L. C. (2005). "Electrical stimulation for wound healing: a review of evidence from in vitro studies, animal experiments, and clinical trials." International Journal of Lower Extremity Wounds 4(1): 23-44.

Kumaran, B. and T. Watson (2015a). "Radiofrequency-based treatment in therapy-related clinical practice – a narrative review. Part I: acute conditions." Physical Therapy Reviews 20(4): 241-254.

Kumaran, B. and T. Watson (2015b). "Thermal build-up, decay and retention responses to local therapeutic application of 448 kHz capacitive resistive monopolar radiofrequency: A prospective randomised crossover study in healthy adults." Int J Hyperthermia 31(8): 883-895.


Kumaran, B. and T. Watson (2015c). "Capacitive resistive monopolar radiofrequency (CRMRF) therapy at 448kHz: localized application significantly enhances and sustains skin physiological responses." WCPT Singapore . Abstract published in Physiotherapy 101: e798.

Kumaran, B. and T. Watson (2015 d). Capacitive Resistive Monopolar Radiofrequency (CRMRF) therapy at 448 KHz: the effects on deep blood flow and elasticity of tissues. PhysiotherapyUK 2015, Liverpool.

Kumaran, B. and T. Watson (2016). "Radiofrequency-based treatment in therapy-related clinical practice – a narrative review. Part II: chronic conditions." Physical Therapy Reviews 20(5-6): 325-343.

Kumaran, B., et al. (2017). "Continuous-mode 448 kHz capacitive resistive monopolar radiofrequency induces greater deep blood flow changes compared to pulsed mode shortwave: a crossover study in healthy adults." European Journal of Physiotherapy: DOI: 10.1080/21679169.2017.1316310

Matsui, Y., A. Nakagawa, Y. Kamiyama, K. Yamamoto, N. Kubo and Y. Nakase (2000). "Selective thermocoagulation of unresectable pancreatic cancers by using radiofrequency capacitive heating." Pancreas 20(1): 14-20.

Ohguri, T., H. Imada, K. Yahara, S. Kakeda, A. Tomimatsu, F. Kato, S. Nomoto, H. Terashima and Y. Korogi (2004). "Effect of 8-MHz radiofrequency-capacitive regional hyperthermia with strong superficial cooling for unresectable or recurrent colorectal cancer." Int J Hyperthermia 20(5): 465-475.

Sakamoto, T., H. Katoh, T. Shimizu, I. Yamashita, S. Takemori, K. Tazawa and M. Fujimaki (1997). "Clinical results of treatment of advanced esophageal carcinoma with hyperthermia in combination with chemoradiotherapy." Chest 112(6): 1487-1493.

Watson, T. (2008a). Electrical Properties of Tissues. Electrotherapy : Evidence Based Practice. Ed: T. Watson. Edinburgh, Churchill Livingstone / Elsevier

Documents

Indiba Summary of the Published and Scientific Evidence...Hepatocarcinoma Cellular Viability (Liver Cancer cells – hepatocarcinoma – HepG2) The aim of this study was to investigate