Patent Application: US-201615213946-A

Abstract:
methods and apparatus for the treatment of dermatological conditions , including , for example , viral infections , microbial infections , cancers , dermatological conditions and particularly infections of the skin caused by human papillomavirus .

Description:
an embodiment of a microwave power generator system for medical applications is illustrated in fig1 the apparatus comprising :— a microwave source for providing microwave energy 1 , connectable to a system controller 2 for controlling at least one property of the microwave radiation provided by the microwave source ; and a monitoring system 3 for monitoring the delivery of energy and an interconnecting cable 4 and an applicator hand piece 5 and a removable applicator means 6 , for example an applicator device , for delivering microwave energy , wherein :— the applicator is configured to deliver precise amounts of microwave energy provided by the source at a single frequency or across a range of frequencies . a typical hpv infection is illustrated in fig2 ( a ) , this comprises the normal tissue 7 , the papilloma surface 8 , the capillary feed network 9 . the terminal differentiation pathway of epidermal cells infected by the hpv virus is illustrated in fig2 ( b ) basal cells 10 become infected with the hpv virus leading to viral replication in the stratum spinosum 11 followed by assembly of virus particles in the stratum granulosum and release of virus particles in the in the stratum corneum ( papilloma surface ). fig3 shows the components of an apparatus according to an embodiment of the present invention , the components shown separately for ease of reference . the apparatus comprises a generator system 14 with a locking microwave connection 15 to a flexible microwave cable 16 connected to a hand piece 17 ( which may have the same type of locking connection ) which accepts an applicator component 19 . the applicator component is designed to match to the tissue properties of the papilloma 20 and not match to the normal tissue 18 . the cable 16 may include both microwave and signal data cables and may be reversible to enable connection to either port . an alternative embodiment of an applicator 21 is illustrated in fig4 with the component having a domed or enclosing surface compatible with raised or curved lesions . the method of inducing a microwave heat shock responses is illustrated in fig5 . in this illustration microwave radiation creates a thermal stress which results in protein denaturation 22 . heat shock proteins ( hsp ) are normally bound to heat shock factors ( hsf ) ( 23 ), but dissociate in the presence of denatured proteins ( pd ). once dissociated , hsps bind to the denatured proteins by rapid release . this requires adenosine triphosphate ( atp ) 24 . further hsps are generated when hsfs phosphorylate ( po4 ) ( 25 ) and trimerize ( 26 ). these trimers bind to heat shock elements ( hse ) 27 that are contained within the promoters of the hsps and generate more protein 28 . newly generated hsps can then free to bind more denatured proteins 29 . the measured dielectric properties for the sample population median versus frequency of plantar verrucae are reported in fig6 ( a ) and 6 ( b ) . measured results for er and loss tangent versus frequency from 2 ghz to 20 ghz are reported . the measurements were made using an agilent pna - l network analyzer connected to an agilent 85070e dielectric measurement system with the 85070e performance probe kit ( option 050 ) measuring from 300 khz to 20 ghz . deionised water and air were used as dielectric references for calibration . the measured dielectric properties for the sample population ( median taken across the population ) versus frequency for various plantar tissues are reported in fig7 . measured results for er versus frequency from 2 ghz to 20 ghz are reported illustrating demarcation between each tissue type . statistically analysed dielectric property values taken over a sample population ( using the median of the measurement range 7 . 5 - 8 . 5 ghz taken from each sample ) for verrucae tissue is presented in fig8 . the median er value was measured at 4 . 93 for this dataset . with reference to fig9 a comparison of statistically analysed measurements of the dielectric properties of various plantar tissues over a sample population ( using the median of the measurement range 7 . 5 - 8 . 5 ghz taken from each sample ) is illustrated . patients with treatment - refractory plantar warts were excluded if they had a pacemaker fitted , were pregnant or breast feeding , had any metal implants within the foot or ankle , suffered any known disease or condition affecting their immune function or their capacity to heal . adverse events were categorized as being specifically associated with the microwave procedure , or unrelated . a complete examination of the affected area was undertaken at each study visit . at the conclusion of the treatment session all patients were given an advice information sheet advised to report any complications . no post - operative dressing was required and patients were advised to subsequently undertake normal everyday activities as usual with no restrictions . a total of 32 patients with 54 foot warts were enrolled into the study . of the 32 , 17 were males and 15 females . ages ranged from 22 - 71 years with a mean age of 44 . 79 years ( sd 13 . 019 ]. of the 54 lesions , 16 were reported as single lesions , and 38 as multiple type lesions ( including mosaic verrucae ). the average lesion duration was 63 months ( 5 . 25 years ) with a range of 2 - 252 months (& lt ; 1 - 21 years ). the mean lesion diameter was 7 . 43 mm ( sd 6 . 021 ), ranging from just 2 mm to 38 mm in diameter . the procedure was performed in an out - patient setting , with standard podiatric facilities . the swift device settings were titrated up as tolerated to 50 j over a 7 mm 2 application area ( 7 . 14 j / mm 2 ). the microwave energy was delivered to the affected area over 5 s duration ( 50 j delivered as 10 watts for 5 s ). lesions which were & lt ; 7 mm in diameter were treated with one application of the probe at a single treatment session whilst lesions & gt ; 7 mm were underwent multiple applications until the entire surface of the wart had been treated . clinical assessments were performed at baseline and at 1 week , 1 month , 3 months , and 12 months after treatment by a podiatrist experienced in the management of plantar warts . response to treatment was assessed by the same investigator as ‘ completely resolved ’ or ‘ unresolved ’. complete resolution was indicated by fulfilling three criteria : i . lesion no longer visible , ii . return of dermatoglyphics to the affected area , iii . no pain on lateral compression . pain was assessed using a 10 point visual analogue scale . normal skin samples were acquired from healthy individuals after obtaining informed written consent with approval by the southampton and south west hampshire research ethics committee in adherence to helsinki guidelines . skin samples were treated immediately ex - vivo with microwave ( swift s800 ; emblation ltd ., uk ) or liquid nitrogen therapy and treated skin excised . excised skin was sent for histological analysis or placed in culture media . histological analysis with hematoxylin and eosin ( h & amp ; e ) tissue sections were undertaken following fixation and embedded in paraffin wax . dna damage was assessed by staining for single stranded and double stranded dna breaks by tunel assay using the apoptag ® in situ apoptosis detection kit ( millipore , uk ). following culture , supernatants were collected and analysed for lactate dehydrogenase release using the cytotoxicity detection kit ( roche applied science ) as a measure of apoptosis . human skin and hacat keratinocytes were cultured in calcium - free dmem ( thermofisher scientific ) with 100 u / ml penicillin , 100 μg / ml streptomycin , 1 mm sodium pyruvate , 10 % fetal bovine serum ( fbs ) and supplemented with calcium chloride at 70 μm final concentration . lymphocytes were cultured in rpmi - 1640 media with 100 u / ml penicillin , 100 μg / ml streptomycin , 1 mm sodium pyruvate , 292 μg / ml l - glutamine , supplemented with 10 % fbs or 10 % heat inactivated human serum ( hs ). hacat cells were cultured at sub - confluency to avoid cell differentiation and used in assays at passage 60 - 70 . cells were plated at 2 . 5 × 103 cells / well in 96 - well flat plate ( corning costar ) and cultured overnight to reach confluence . hacats were washed once with pbs before treatment with 150 j microwave , liquid nitrogen ( 10 s ), heat ( 42 ° c . preheated media ) or with lps + ifn - γ ( 1 ng / ml + 1000 u / ml ). cells were cultured for 24 h before supernatants were harvested . for hpv - specific t cell lines , pbmcs were isolated from hla - a2 individuals as previously described 11 . pbmcs were seeded at 2 - 4 × 106 cells / well in 24 - well culture plate and 10 μg / ml of 9 mer hla - a2 restricted hpv16 epitope llm ( llmgtlgiv ) 12 was added , cells were cultured in 1 ml rpmi + 10 % hs . on day 3 , cells were fed with rpmi + 10 % hs + il - 2 ( 200 iu / ml ), and then fed again on day 7 or when needed . after day 10 , hpv - specific t cells were harvested for cryopreservation before testing against hpv in elispot assays . monocyte derived dendritic cells ( modcs ), cd14 + cells were positively isolated from pbmcs by magnetic separation using cd14 microbeads ( milentyi biotec ), according to manufacturer &# 39 ; s protocol . cells were washed and resuspended in rpmi + 10 % fbs + 250 u / ml il - 4 and 500 u / ml gm - csf . at day 3 , cells were fed with rpmi + 10 % fbs + il - 4 and gm - csf , and then harvested on day 5 for use in functional assays . in vitro , microwave therapy of cell cultures was delivered through the base of the plastic culture dish and showed a linear dose response between the energy delivered and thermal induction ( not shown ). utilising the equation e = m × c × θ ( e = energy transferred , j ; m = mass , kg ; c = specific heat capacity , j / kg ° c . ; θ = temperature change , ° c . ), we calculated that in our system the 150 j swift programme delivered 15 . 58 j ( s . d . 0 . 921 ) through the plastic to the culture . keratinocytes were treated with microwave at various energy settings before removal of supernatant at various time points . modcs were treated overnight with keratinocyte supernatant , then washed twice before incubation with 10 μg / ml llm peptide for 2 hours before a further wash . human ifn - γ elispot ( mabtech , sweden ) was undertaken as per manufacturer &# 39 ; s protocol and as reported previously 11 . 1 × 103 modcs were plated with autologous hpv peptide - specific t cells at 1 : 25 ratio . spot forming units ( sfu ) were enumerated with elispot 3 . 5 reader ( aid , germany ). modcs were treated with hacat supernatant and harvested at 24 hours for flow cytometric analysis of cell phenotype . cells were stained with violet live / dead stain ( invitrogen ) for 30 min at 4 ° c ., then washed with pbs + 1 % bsa and stained with antibodies percp - cy5 . 5 anti - hla - dr , fitc anti - cd80 , fitc anti - cd86 , pe anti - cd40 , all purchased from bd , for 45 min at 4 ° c . cells were washed then resuspended in pbs + 1 % bsa and analysed using the bd facsaria and the flowjo v10 . 0 . 08 analysis software . the expression of chosen genes was validated with quantitative pcr , using the taqman gene expression assays for target genes : ywhaz ( hs03044281_g1 ), irf1 ( hs00971960_m1 ), irf4 ( hs00543439_ce ) ( applied biosystems , life technologies , paisley , uk ) in human skin treated as indicated . rna extraction ( rneasy micro kit , qiagen ) and reverse transcription ( nanoscript kit ; primer design , southampton , uk ) were carried out accordingly to the manufacturer &# 39 ; s protocol . treatment of human papilloma virus infection in humans with microwave therapy from january 2015 to september 2015 at the university of southampton , we enrolled 32 patients with severe , treatment - refractory plantar warts . the diagnosis of plantar wart was confirmed by a podiatrist experienced in management of such lesions . a clinically significant wart was defined as & gt ; 1 year duration , which had failed at least two previous treatments ( salicylic acid , laser , cryotherapy , needling and surgical excision ). in each patient , the most prominent plantar wart ( most severely affected ) was targeted for treatment ( fig1 a and 10b ). at the end of the study period , of the 54 warts treated , 41 had resolved ( 75 . 9 %), 9 remained unresolved ( 16 . 7 %), 3 warts ( n = 2 patients ) had withdrawn from the study ( 5 . 6 %) and 1 patient ( with 1 wart ) was lost to follow up ( 1 . 9 %). the mean number of days to resolution 79 . 49 days ( sd 34 . 561 ; 15 - 151 days ). 94 % of resolving lesions had cleared after 3 treatments ( fig1 c ). no significant difference in resolution rates were observed between males and females ( p = 0 . 693 ) was observed . human skin has not been previously treated with microwave therapy , therefore , we proceeded to undertake a full histological analysis . skin removed during routine surgery was sectioned 1 hour after treatment ex vivo . neither macroscopic , nor histological changes were noted with the lowest energy setting ( 5 j ). at 50 j , mild macroscopic epidermal changes only were noted , and microscopically minor architectural changes , and slight elongation of keratinocytes were seen without evidence of dermal collagen sclerosis . at higher energies ( 100 , 200 j ) gross tissue contraction was visible macroscopically . microscopic changes in the epidermis were prominent , showing spindled keratinocytes with linear nuclear architectural changes and subepidermal clefting ( fig1 a ). dermal changes were prominent at energies of 100 j and above and showed a homogenous zone of papillary dermal collagen , thickened collagenous substances , accentuation of basophilic tinctorial staining of the dermal collagen with necrotic features ( fig1 a ). these features are similar to electrocautery artefacts and suggest the potential to induce scarring at & gt ; 100 j . histological analysis both at 16 h and 45 h showed similar changes ( not shown ). in clinical practice , cryotherapy is delivered to the skin by cryospray , which is time - regulated by the operator . in contrast to microwave therapy , minimal epidermal or dermal architectural change was identified with cryotherapy at standard treatment duration times ( 5 - 30 s ), but did show a dose dependent clumping of red blood cells in vessels ( fig1 b ). tissue release of ldh acts as a biomarker for cellular cytotoxicity and cytolysis . to examine the extent of cell death induced by microwave irradiation , human skin was treated with 0 , 50 , 100 or 200 j before punch excision of the treated area and incubation in medium for 1 hour or 16 hours . measurement of ldh revealed a dose dependent induction of tissue cytotoxicity with increasing microwave energies ( fig1 c ). in line with the lack of histological evidence of cellular damage , at 5 j , cytotoxicity of microwave application was equivalent to control . early cytotoxicity was not prominent at 50 j , but became more evident after 16 hours . higher energy levels induced more prominent cytotoxic damage . in contrast to microwave therapy , liquid nitrogen treatment of skin induced cytotoxicity at the lowest dose both at 1 hour and 16 hours . terminal deoxynucleotidyl transferase dutp nick end labelling ( tunel ) identifies cells in the late stage of apoptosis . analysis at 0 , 5 , 50 , 100 and 200 j identified increased cellular apoptosis in the epidermis above 100 j ( fig1 a ). in contrast , cryotherapy with a liquid nitrogen spray applicator directly to the skin as used in clinical practice , even at a very short treatment time ( 5 s ) induced significant epidermal and dermal dna fragmentation ( fig1 b ). the physics of microwave therapy suggests a tight boundary between treated and untreated tissue with minimal spreading of the treated field . this was borne out histologically by a clear demarcation between treated areas extending vertically from the epidermis through the dermis ( fig1 d ). examination of the dermis showed that microwave therapy modified skin adnexae inducing linear nuclear architectural changes in glandular apparatus , microthrombi , fragmented fibroblasts and endothelial cells ( fig1 e ). we first examined the response of keratinocytes to microwave therapy in vitro . keratinocyte apoptosis was induced by microwave therapies above 100 j in vitro ( fig1 a ). only above the apoptotic threshold were surface phenotypic changes of cellular activation noted in viable cells with increased expression of hla - dr , cd40 and cd80 ( fig1 b ). next , we utilised a model of skin cross talk of keratinocyte signalling to dermal dendritic cells . keratinocytes were treated with microwave therapy as above , and washed after 8 hours to remove dead or apoptotic cells . treated keratinocytes were then incubated for a further 16 hours before supernatant collection to prime monocyte derived dendritic cells ( modcs ) which had not been directly exposed to microwave therapy . this showed a potent induction of modc activation with increased expression of cd86 , cd80 and to a lesser extent cd40 ( fig1 c ). we next set out to test the functional outcome on skin dendritic cells following microwave treatment of keratinocytes . keratinocytes were untreated , microwave , or cryotherapy treated before supernatant harvesting . supernatant primed dcs were pulsed with a 9 amino acid hla - a2 epitope ( llm ) from human papilloma virus ( hpv ) e16 protein and cultured with an autologous hpvspecific cd8 + t cell line . as expected , in all conditions , the dcs efficiently presented hpv peptide to cd8 + t cells inducing ifn - γγ ( fig1 a ). however , dendritic cell presentation of hpv virus is dependent upon cross - presentation to the mhc class i pathway . therefore we also tested the ability of untreated , microwave treated or cryotherapy kc - primed dcs to present human papilloma virus ( hpv ) e16 protein to an hla matched hpv - specific cd8 + t cell line . strikingly , only microwave treated kcs were able to prime dcs to enhance cross - presentation ( fig1 b ). to explore the potential mechanism of keratinocyte response to microwave therapy we confirmed up regulation of hsp - 70 in response to microwave therapy of keratinocytes ( fig1 c ) and showed significant il - 6 induction in keratinocytes above that seen in cryotherapy treated cells ( fig1 d ). irf1 and irf4 are key regulators of dendritic cell activation and we confirmed that microwave therapy induced down regulation of irf1 and up - regulation of irf4 ( fig1 e ). this is the first study to investigate the potential efficacy of locally delivered microwaves in the treatment of cutaneous viral warts in vivo . we report a complete resolution rate of 75 . 9 % recalcitrant plantar warts ( average lesion duration of over 5 years ). this compares very well with previous reports of plantar wart resolution for salicylic acid and or cryotherapy ( 23 - 33 %) 13 . whilst this study was a pilot phase , and did not include a control untreated arm , we believe the treatment effect to be significant . for all novel therapies , adverse events are critical . in this study we did not identify a strong signal for adverse events with microwave therapy of cutaneous warts . as with current physical treatments for warts discomfort is expected for the patient . during the study patients typically reported that for a typical 5 second treatment that they endured moderate discomfort for approximately 2 seconds , which immediately diminished after the treatment had completed . in addition , it was commonly noted that discomfort was less with subsequent treatments . one male patient , withdrew from the study after one treatment , citing the pain of treatment as the reason . in the study design phase , preoperative use of topical anaesthetic cream was tested , but appeared to do little to mitigate the pain ( unpublished data ) and it was felt that the pain of local anaesthetic injection would exceed that normally experienced during a microwave treatment . following microwave therapy , patients did not require dressings or special advice as microwave therapy utilised in this study did not cause a wound or ulcer in the skin , allowing the patient continue normal activity . the short microwave treatment time ( 5 s ) offers a significant clinical advantage over current wart therapies such as cryotherapy and electro - surgery . within 5 s , microwaves penetrate to a depth of over 3 . 5 mm at the energy levels adopted for the study 14 — possibly a greater depth than can be attained by cryosurgery or laser energy devices . moreover , as microwaves travel in straight lines energy is deposited in alignment the device tip with little collateral spread , meaning minimal damage to surrounding tissue , as observed in this study . microwaves induce dielectric heating . when water , as a polar molecule , is exposed to microwave energy , the molecule is excited and rotates attempting to align with the alternating electromagnetic field . at microwave frequencies the molecule is unable to align fully with the continuously shifting field resulting in heat generation . within tissues , this acts to rapidly elevate temperatures . this process rapidly changes cellular heat because it does not depend on tissue conduction . microwave treatment produces no vapour or smoke unlike ablative lasers and electro - surgery , eliminating the need for air extraction systems due to the risk of spreading viral particles within the plume 15 . although , microwave therapy has been considered a tissue ablation tool , we saw minimal skin damage after treatment with 50 j , yet apparent good clinical response . therefore we investigated whether there was evidence to support an induction of immunity by microwave therapy . the critical nature of cd8 + t cell immunity for host defense against hpv skin infection is well established and supported by the observation of increased prevalence of infection in immunosuppressed organ transplant recipients 16 , and that induction of protection from hpv vaccines is mediated by cd8 + t cells 17 . we show here , that microwave therapy of skin induces keratinocyte activation and cell death through apoptosis . however , at sub - apoptotic doses , microwave primed keratinocytes are able to signal to dendritic cells and enhance cross - presentation of hpv antigens to cd8 + lymphocytes which offers a potential explanation for the observed response rate in our clinical study . in vitro evidence suggests that this is likely to be mediated by cross - talk between microwave treated skin keratinocytes and dendritic cells , with resultant enhanced cross - presentation of hpv protein to cd8 + t cells . microwave therapy also induced enhanced il - 6 synthesis from keratinocytes . il - 6 , is a pro - inflammatory mediator , important in anti - viral immunity which has been recently shown to induce rapid effector function in cd8 + cells 18 . thus , il - 6 up - regulation may provide an important additional mechanism for microwave anti - viral immunity . irfs have been shown to be central to the regulation of immune responses 19 - 21 . irf4 is essential for differentiation of cytotoxic cd8 + t cells 22 23 , but up - regulation in dendritic cells has also been shown to enhance cd4 + differentiation , thereby potentially enhancing both cd8 + immunity and t cell help following microwave treatment . irf1 expression has been previously reported to be modulated by hpv infection , but different models have shown opposite outcomes 24 , 25 . we show down - regulation of irf1 in human skin in association with a microwave therapy which supports the proposal of irf - 1 as a therapeutic target in hpv infection 28 . this study is the first of its kind studying microwaves in the treatment of plantar warts in vivo . however , the authors acknowledge the limitations of the uncontrolled , non - randomised design . despite the promising results shown here , studies with larger sample sizes are needed to assess the efficacy of this treatment and for infrequent but serious adverse events . it will be understood that embodiments of the present invention have been described above purely by way of example , and modifications of detail can be made within the scope of the invention . each feature disclosed in the description , and ( where appropriate ) the claims and drawings may be provided independently or in any appropriate combination . 1 . cockayne s , hewitt c , hicks k , et al . cryotherapy versus salicylic acid for the treatment of plantar warts ( verrucae ): a randomised controlled trial . bmj 2011 ; 342 : d3271 . 2 . stern p l . immune control of human papillomavirus ( hpv ) associated anogenital disease and potential for vaccination . journal of clinical virology : the official publication of the pan american society for clinical virology 2005 ; 32 suppl 1 : s72 - 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