VOLUME 1 ISSUE No. 2SPRING 2013IJOP

The International Journal Of Phytotherapy

Development of Blood TestsFor Early Cancer DetectionBrian Schaefer, D.Phil. (Oxon)

Going Back To Our RootsThis months featured herb is the dandelion (Taraxacum Officinale)

Reishi Mushroon Active IdentifiedPhytotherapy News

THIS ISSUE

Development of Blood Testsfor Early Cancer Detection

To study the prevalence of sedentary behavioursand decreased physical activity amongschool going adolescents in Ludhiana

Reishi Mushroon Active Identified

Reishi mushrooms have been used in traditionalchinese medicine for thousands of years

COVER STORY

PHYTOTHERAPY NEWS

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IJOPThe International Journal Of Phytotherapy

The development of these blood tests for earlycancer detection and monitoring build upontwo central bodies of research – research intothe anti-cancer activities of the phytonutrientsknown as Salvestrols and the research into themetabolic activity of the enzyme CYP1B1

1. Sanjeev Kumar Khanna. Assistant ProfessorCollege of Physiotherapy,Christian MedicalCollege & Hospital,Ludhiana

2. Avnee Sarin Intern,College ofPhysiotherapy, Christian Medical College &Hospital,Ludhiana

3. K.E.Benjamin . Vice Principal,College ofPhysiotherapy, Christian Medical College &Hospital,Ludhiana

EDITORIAL

Ginseng Research

Phytotherapy Research News

Panax Ginseng is one of the mostextensively studied medicinal plants, andthe roots of ginseng contain a multitudeof bioactive molecules each with theirown pharmacological action. The mainbioactive compounds are triterpeneglycosides known as ginsenosides. Thereis a mysterious duality to the actions ofginseng which are likened to a ying yangof opposites. On the one hand ginsengcan help with wound healing whichinvolves angiogenesis, and on the otherhand ginseng has anticancer effects dueto its anti-angiogenic action. How can oneplant have these two opposite actions.Well it turns out there are two differentmolecules responsible for these actionsone of which is pro angiogenic and theother is anti-angiogenic. These are bothginsenosides of the panaxadiol family anddiffer mainly in the positions of theglucose goups. The pro angiogeniccompound is ginsenoside Rg1 which is apanaxatriol glycoside, and works bybinding to a human glucocoticoidreceptor to elicit an angiogenic responseand aid in wound healing.

The anti-angiogenic compound has beenidentified as ginsenoside Rb1 which is apanaxadiol glycoside, and this works bybinding to the human estrogen receptorER-beta (but not ER-alpha) where it actsas an agonist to promote ER-beta DNAtranscription and protein expression. Thisleads to the ER-beta stimulatedproduction of the powerful natural anti-angiogenic protein PEDF. This is the mostpotent anti-angiogenic protein expressedby the human body and so the Rb1stimulated production of PEDF accountsfor its antiangiogenic activity. Thisreasearch shows the importance of theglucose carbohydrate groups in elicitingbiological function. However theginsenoside glucosides are modified byintestinal bacterial action which involvesthe sugar moieties being removed. Theginsenoside Rb1 is a tetra-glucosederivative that is cleaved to the mono-glucose derivative compound K that is theactive metabolite that passes through theintestinal wall and enters thebloodstream. What is interesting here isthat the ginsenoside mono-glucoseconjugate is able to be absorbed with thesugar molecule intact into thebloodstream and this is probably due tothe high lipophilicity of the panaxadioltriterpene group.

Reishi Mushroon Active Identified

Reishi mushrooms have been used intraditional chinese medicine forthousands of years and are among themore popular alternative treatments forcancer. Even though the anticanceractivity of Reishi mushrooms isacknowledged by alternative therapypractitioners it has taken until now forscientists to identify the activecomponent. Recent research has lead tothe identification of the active compoundGanoderic Acid, named after the Reishimushroom genus Ganoderma Lucidum.Ganoderic Acid is an oxidised derivativeof the triterpene lanosterol and is thefungal metabolite that has resulted fromlanosterol oxidation. This compound hascarbonyl goups at the 3 and 10 positionsof the steroid which mimic cortisone.Ganoderic Acid has been found to inhibitthe enzyme Topoisomerase which is thetarget of the clinically used anticanceragents Topotecan and Irinotecan.

Development of Blood tests for EarlyCancer Detection

The main feature article of this edition ofIJOP describes research aimed atdeveloping a test for cancer that willallow much earlier diagnosis than ispresently available. The article describestwo new cancer diagnostic blood tests.The first directly measures the levels ofthe CYP1B1 protein in the bloodstream.In normal volunteers the level of CYP1B1is vanishingly small but this level is highlyelevated in cancer patients which makesit useful for early cancer diagnosis. Thesecond test measures the salvestrolmetabolites that result from CYP1B1activity. The levels of unreactedsalvestrol and activated salvestrol

metabolites are measured in thebloodstream and this gives an indicationof how well the salvestrols are working byproviding evidence for their metabolism.

This months IJOP includes a guest articlefrom the College of Physiotherapy in thecity of Ludhiana in Northern India. Thisarticle presents evidence for aconcerning trend in increased sedentarybehaviour of adolescents in India, andshows that the developing world isfollowing the western world in this trendwhich can lead to an array of healthproblems later in life.

Professor Gerry PotterProfessor Gerry Potter

Editor

FEATURED HERB

Featured Herb – Dandelion

Going Back To Our Roots

This months featured herb is thedandelion (Taraxacum Officinale) which ispart of the Compositae family of plants.This family has two sub-families known bytheir trivial names as the “Daisy Family”which includes many yellow floweredplants such as sunflower, yarrow andtansy, and the “Thistle Family” which arepurple flowered and includes milk thistle,burdock and artichoke.

The diuretic properties of dandelionleaves are well known historically but ittook until 1978 for this effect to beclinically proven. Dandelion has beenused traditionaly to treat various diseasesincluding cancer, and one of its traditionalnames is “Cankerwort”. Phase 1 clinicaltrials of dandelion root tea for treatingcancer have just started in Canadafollowing observations of its beneficialactivity. This is an importantdevelopment and is one of only a fewexamples of a natural product enteringclinical trials.

There are 3 classes of molecules indandelion that have anticancerproperties, the sesquiterpene lactones,the triterpenes, and the salvestrols. Thesesquiterpene lactones such as taraxinicacid are toxic compounds and are theplants “don't eat me” molecules. Theseare the anti feedants that deter grazinganimals from eating them. The lactonering contains an acrylate group which isan alkylating agent that can react withproteins and DNA bases causing toxicity.In this respect they are similar to theanticancer alkylating agents used in

chemotherapy such as Melphalan. Thesemolecules are mopped up by glutathionein the liver and these molecules elicit adetoxification response which involvesexpression of glutathione by the liver andincreased urination resulting in itsdiuretic effects. These molecules aremainly present in the leaves and givesthem their characteristic bitter taste. Theyoung leaves have lower levels and canbe safely eaten and added to green salads.

As a food dandelion leaves and roots area source of vitamins, minerals andcarbohydrate. The vitamins present arepro-vitamin A (carotene), vitamin B3(niacin) and vitamin C. The mineralspresent include potassium, calcium andmagnesium. The carbohydrates presentare short polymers of glucose andfructose in the form of Inulin. It isinteresting that dandelions contain boththe salvestrols and their co-factors suchas niacin and magnesium which arerequired for their biological activation, somother nature has formulated all thesecomponents together in a single plant.

The triterpenes found in dandelioninclude lupeol, taraxasterol, beta-sitosterol, and stigmasterol. These aremade by the plants using a complexsynthetic route involving squalenecyclisation, by a process similar to thatused by the human body to make thehuman triterpenes lanosterol andcholesterol. These plant derivedtriterpenes can interfere with cholesterolbiosynthesis and have the ability to lowercholesterol levels. Lupeol has recentlycaught the attention of researchers sinceit is able to induce apoptosis in tumourcells without causing normal cell toxicityin a similar manner to the salvestrols.Lupeol is also found in exotic fruits suchas mangoes. Lupeol is related to betulinfound in the silver birch that also haspro-apoptotic activity. The triterpenesand salvestrols are found mainly in thedandelion roots and are present as their

sugar glycoside derivatives.

A fourth class of molecules is present indandelions that have anti-inflammatoryactivity which are the phenolic acidgylcosides such as Taraxacoside. This is aglucose conjugate of 4-hydroxyphenylacetic acid which is similar to thenon steroidal anti-inflammatory drugsDiclofenac and Ibuprofen.

Dandelion roots are a rich source ofsalvestrols and these are now thought tobe the principal contributors to itsanticancer activity. The dandelionfeatures in traditional herbal medicinemanuscripts and is found in the WelshHerbal written in the 12th century.Culpepper describes dandelion as goodfor increasing urinary flow, and forhealing inner ulcers. Chevallier's

“Encylcopedia of Medicinal Plants”describes dandelion root as havingpowerful anti-inflammatory propertiesuseful for the treatment of eczema,psoriasis, and arthritis, and these are allconditions which can respond totreatment with salvestrols. In traditionalchinese medicine (TCM) dandelion root isknown as Pu Gong Ying and has beenused in China for breast cancer therapy.

I like the dandelion and admire itsresilience and ability to survive in the wild.It has a cheerful yellow flower that liftsthe spirits and brightens up our lawns.They do not need watering or fertilizersand happily grow without any humanassistance. They are a gift from mothernature which are freely available and arean important component of the herbalmedicine cabinet.

Recipe for Dandelion & Burdock TeaTake 5 tsp of dandelion root and 2 tsp ofburdock root and place in a saucepan.Add 250 ml of boiling water and simmerfor 20 mins. Allow to cool then strainthrough a sieve into a jug ready to drink.

ABSTRACTObjective: To study the prevalence of sedentary behaviours and decreased physical activity among school going

adolescents in Ludhiana.Material and Method: School going children of Ludhiana of age group 13-17 years were enrolled in the presentstudy. All subjects were given a Questionnaire for this survey which was filled and completed by them and their

leisure time spent in sedentary and non-sedentary behaviour per day during weekday and weekends wascollected and then collected data was analysed.

Result: 100 school going students of Ludhiana, between age group of 13-17 years were taken under theinclusion criteria. The result of the study showed that the students spent 266 minutes (4.43 hours) on average

in sedentary activity and only 15 minutes were spent in non – sedentary behaviours on weekdays and onweekends time spent on sedentary activity was increased to 275 minutes (4.58 hours)in sedentary activity and

15 minutes in non- sedentary one.Conclusion: This survey has concluded that there is prevalence of sedentary lifestyle and decreased physicalactivity among school going adolescents. Our sample showed that there was very low level of leisure timephysical activity among school going adolescents as recommended by W.H.O. So, there is major need to

promote leisure time physical activity among adolescents to have a better health in future, prevent obesity andother health related problems.

Keywords: sedentary lifestyle, non- sedentary lifestyle, Ludhiana school going adolescents, leisure timesedentary activity questionnaire.

UPFRONT

obesity, the number of cardiovasculardiseases and is positively associated with

physical fitness.[4] Furthermore, there arestrong evidence that sedentarybehaviours adopted during childhood

track into adult life.[5]

Recommended guidelines for physicalactivity and time spent in other sedentaryactivities say that children and youthshould accumulate at least 60 minutes of

physical activity on a daily basis.[6]

Extended period of time (2 hours or moreper day) spent on sedentary pursuits ( TVviewing, computer use , etc.) areassociated with decreased physicalactivity levels and increased obesity and

overweight.[6]

The present study, helps us to knowindividual students leisure time sedentaryand physically active behaviours.Materials and Methodology-It was a Survey study conducted on aschool students with total sample size of100, between age group of 13-17 years.

Stratified Random Sampling techniquewas used.

HypothesisNull Hypothesis: The Children haveadequate amount of Physical Activity andless sedentary behaviour.Alternate Hypothesis: The Children havedecreased level of Physical Activity andmore sedentary behaviour.Variables:Duration of participants physical activities(sports, active transport and otherhabitual physical activity) and sedentaryactivities (TV viewing, computer use sitand talk etc) in leisure time was convertedinto minutes.Instruments:Leisure time physical activityquestionnaire for adolescents .Procedure-Firstly, questionnaire was prepared whichincluded close ended questions only. Thena pilot study was done on 20 students to

BACKGROUNDA sedentary lifestyle is the medical termused to denote a type of lifestyle with no

or irregular physical activity.[1][2]

Sedentary activities include sitting,reading, playing certain videogames, usingthe computer, watching television, etc. formuch of the day with little or no vigorous

physical exercise.[2]

Physical activity is a fundamental meansof improving physical and mental

health.[3]

Adolescence is the period of psychologicaland social transition between childhoodand adulthood. The health habits andcoping skills developed during this periodcarry into adulthood. Adopting adequatephysical activity levels during childhoodand adolescence is important to prevent

chronic disease later in life. [4] Regularparticipation in physical activity is anessential component of a healthy lifestyle.[4] Among adolescents regular physicalactivity actually reduces the onset of

To study the prevalence of sedentary behavioursand decreased physical activity amongschool going adolescents in Ludhiana

By Sanjeev Kumar Khanna.[1] Avnee Sarin [2] K.E.Benjamin .[3]

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weekdays than weekends i.e. onweekdays average 42 minutes spent perday.The least time was spent on playing sports,outdoor games, exercises, other physicalhobbies and active transport.

Fig: 2 b Shows total screen time was 133minutes (~2hours) per day and 161

check the validity and reliability of thequestionnaire by intertester andintratester methods. Questions wereeasily understood and well answered bysubjects.After the successful completion of pilotstudy the questionnaire was distributedamong 100 students. Each subject is givenverbal introduction for the questionnaire.A signed consent form was obtained fromeach subject prior to participation in thestudy. After this subjects were made tofill the explained questionnaire and aregiven instructions to tick the option whichmost closely relates them. After gettingback the Questionnaires data wasanalysed and results were obtained.

DATA ANALYSIS: Data was analysed inMicrosoft excel 2007 by calculatingaverage, percentage, mean, median andmode method and final results are formedwhich are as under pie graph 1 and 2.Table 1 shows the average mean timespent in leisure sedentary and non-sedentary behaviour during weekdays andweekends.

FIG: 1 a (on the next page) Shows averagetime spent on sedentary activity was 266minutes (4.43 hours) per day with +/-63.3SD and 275 minutes (4.58 hours) per day+/- 69.8 SD weekdays and weekendsrespectively.Time spent on non-sedentary activity was15 minutes per day +/- 10.2 SD onweekdays and 15 minutes +/- 15 SD onweekends.TABLE: 2a shows per question the averagemean time spentFig: 2 a Shows students leisure time spenton different activities both on weekdaysand weekends. The graph shows doinghomework, taking tuitions and readingbooks occupied most of the leisure timei.e. average 90 minutes(1hour 30 minutes)per day both on weekdays and weekends.After this T.V. viewing, listening to music,sitting and talking with family and friendswas the most common activity.The other commonly done activity wasusing computer and playing videogamesand other sedentary activities of arts. Theaverage time shows that using computer,playing videogames and T.V. viewingactivity was more on weekends than thatof weekdays.The graph also showed that usage ofmotorized transport was more on

PIE GRAPH: 2

PIE GRAPH 1

Table 1

minutes (~ more than 3hours) per day onweekdays and weekends respectively.

DISCUSSIONThis study reports descriptive data onphysical activity and sedentary behavioursof adolescents. Using questionnairemethod enabled us to get information ontime spent on various activities duringleisure time.In our sample as in other data sets from

Australia7, USA8, UK9-10, Hungary11 and

elsewhere12, TV viewing was the mostpopular sedentary behaviour in leisuretime. Estimates from the present sample

are similar to those from other studies12,with more than 1hours of TV viewing forweekdays and above 2 hours on weekends.There is broad agreement that 4 hours of

TV viewing per day is excessive13 and that< 2 hours per day are recommended. Withthe development of new technologiesscreen time should be focused (TV,computer, videogames etc.). In our studyin addition to TV viewing students spent~1hour on weekdays using computer andplaying videogames and >1hour onweekends. This appears to be similarestimates for other countries when

averaging across a 7 day week.12

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their time may be spent at home. Timeinside home is predictive of moresedentary behaviour.Alongside sedentary behaviours, themethod adopted in this study allowed usto estimate time in more activebehaviours. Students in our sampleappeared to have a very low levels ofleisure time physical activity in the formof sports/ exercise averaging on 14-15minutes weekdays and 10-12 minutesduring weekends, which is much less.In addition to targeting education insedentary behaviour our data suggest thata major effort is needed to promoteleisure time physical activity amongadolescents, whether this be throughsports, formal exercise or informal formsof play etc.This study has provided useful explanatoryand descriptive data on sedentary andactive behavioural pattern of leisure time

use of adolescent students of Ludhiana inIndia.CONCLUSIONThis survey has concluded that there isprevalence of sedentary lifestyle anddecreased physical activity among schoolgoing adolescents. Our sample showedthat there was very low level of leisuretime physical activity among school goingadolescents as recommended by

W.H.O,[19] which states children andyouth should accumulate in at least 60minutes of physical activity per day. So,there is major need to promote leisuretime physical activity among adolescentsto have a better health in future, preventobesity and other health related problems.

FUTURE SCOPEThis study has provided useful explanatoryand descriptive data on sedentary andactive behavioural patterns of leisure timeuse of the adolescents. Such data mayprove useful in gaining a more detailedunderstanding of sedentary and activebehaviours for future interventions,including better health educationpromotion as well as monitoring of trends

.Moreover this study can be done on alarger sample size and in different schoolsin different areas to know the exactsituation in strata.

LIMITATIONSThe survey is limited by sample size of thesubjects which was restricted to 100. Thesurvey was carried out among schoolgoing adolescents of Ludhiana, if it hadbeen done in other schools or in a widerarea, the results might have been different.

FIG: 1

Table 2 A

This total screen time for weekdays in thesample was approximately 2.2 hours. andfor weekends 3 hours or more per day. Soit is important to help young peoplemonitor the amount of their time spenton screen behaviours as some will behaving excessive levels. The data suggeststhat prolonged sitting time, typical ofscreen use may be associated with

unfavourable metabolic profiles.14 So,

breaks in the sedentary time15 and moretime in moderate or moderate to vigorousphysical activity are recommended.At the same time our data showed thatnon screen based sedentary behavioursare also prevalent. These includemotorized transport, doing homework(most common) etc. This suggests higher

levels than in some other countries.16

Scully and colleagues,17 for examplehighlighted that Australian stateeducation department guidelines suggest

, that students complete about 10 minutesof homework a day each school year, theyprogress in school to a minimum of 2hours per day in year 12. Homeworktherefore is a significant sedentary pursuit.Being sedentary for transport is one areawhere policy and promotion can beeffective. There is clear competitionbetween active and motorized modes oftransport. For students living close to theirschool there is good opportunity to takemore active forms of transport and tooffset the decline in walking and cycling

that is seen in many countries.18 Forexample, in our sample 80% of thestudents take no active transport atweekends, suggesting that a great deal of

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9. The Henry J. The role of media inchildhood obesity. Kaiser FamilyFoundation 2004;February 1-1210. Biddle SJH, Gorely T, Marshall SJ, et al.The prevalence of sedentary behaviourand physical activity in leisure time: astudy of Scottish adolescents usingecological momentary assessment.Prevent Med 2009;48:151–5.11. Gorely T, Marshall SJ, Biddle SJH, et al.The prevalence of leisure time sedentarybehaviour and physical activity inadolescent girls: An ecological momentaryassessment approach. Int J Pediat Obes2007;2:227–3412. Pal Hamar, Stuart Biddle, Istvan Soos ,Bence Takacs, Agnes Huszar , Theprevalence of sedentary behaviours andphysical activity in Hungarian youth.European Journal of public health ; Vol.20,no. 1, 85-90.13. Marshall SJ, Gorely T, Biddle SJH. Adescriptive epidemiology of screenbased

media use in youth: a review and critique.J Adoles 2006; 29:333–49.14. Elk Grove Village III ,AmericanAcademy of Pediatrics. Television and thefamily: American Academy of Pediatrics;198615. Hamilton MT, Healy GN, Dunstan DW,et al. Too little exercise and too muchsitting: Inactivity physiology and the needfor new recommendations on sedentarybehavior. Curr Cardiovas Risk Rep-2008;2:292–8.16. Healy GN, Dunstan DW, Salmon J, etal. Breaks in sedentary time: Beneficialassociations with metabolic risk. Diab Care2008;31:661–6.17. Biddle SJH, Gorely T, Marshall SJ, et al.The prevalence of sedentary behaviourand physical activity in leisure time: astudy of Scottish adolescents usingecological momentary assessment.Prevent Med 2009;48:151–5.18. Scully M, Dixon H, White H, et al. Diet,physical activity and sedentary behaviouramong Australian secondary students in2005. Health Promot Int 2007;22:236–45.

19. WHO (2011); Global recommendationson physical activity for health. On – line.Available at:[http://www.who.int/dietphysicalactivity/pa/en/index.html]

Fig 2 A

Fig 2 B

REFERENCES

1. Prevlance of sedentary lifestyle, Centrefor Disease Prevention19912. Foster C. Guidelines for health-enhancing physical activity promotionprogrammes. The European Network forthe Promotion of Health-EnhancingPhysical Activity. Tampere, the UKKInstitute for Health Promotion Research,20003. Physical activity and health in Europe:evidence for action / edited by Nick Cavill,Sonja Kahlmeier and Francesca Racioppi. WHO Library Cataloguing in PublicationData(2006)4. Trost S.G., Saunders, R., Ward D.S.(2002). Determinants of physical activityin middle school children. AmericanJournal of Health Behaviour; 26(2):95-102.5. Louise L. Hardy, PhD, Anthony D. Okely,PhD, Michael L. Booth, Sedentariness,Small- Screen Recreation, and Fitness inYouth. American journal of PreventiveMedicine; 20096. Bates, Heidi. Daily Physical Activity forChildren and Youth: a review and synthesisof the literature, Alberta: ISBN 0-7785-4751-5.,GV443.B329;2006, 613.704 27. Pal Hamar, Stuart Biddle, Istvan Soos ,Bence Takacs, Agnes Huszar , Theprevalence of sedentary behaviours andphysical activity in Hungarian youth.European Journal of public health ; Vol.20,no. 1, 85-90.

8. Olds T, Ridley K, Dollman J.Screenieboppers and extreme screenies:the place of screen time in the timebudgets of 10–13 year-old Australianchildren. Aus New Zeal J Pub Health2006;30:137–42.

Development of Blood Tests for Early Cancer DetectionBy

Brian Schaefer, D.Phil. (Oxon)CARE Biotechnologies Inc.

AbstractCARE Biotechnologies Inc is a biotechnology company formed to initially build upon the

knowledge obtained through the program of research and development of Salvestrols andCYP1B1 and extend that knowledge to development of clinical tools for the detection andmonitoring of cancer. This article is intended to provide the reader with an inside look atthe process of translating this understanding into clinical tools, the challenges faced and

the progress that has been made.

IntroductionThe development of these blood tests forearly cancer detection and monitoringbuild upon two central bodies of research

– research into the anti-cancer activities ofthe phytonutrients known as Salvestrolsand the research into the metabolicactivity of the enzyme CYP1B1. Many ofyou will be familiar with Salvestrols. Forthose that are not Salvestrols are food-based, secondary plant metabolites thathave a specific functional relationship toa cytochrome p450 enzyme calledCYP1B1. CYP1B1 is a unique cytochromep450 in that it doesn’t occur in healthytissue – it only occurs in cancer cells. It isalso unique in that it is a universal cancermarker occurring in every cancer tested.

What the research team discovered is thatSalvestrols work as natural prodrugs.They are exceptionally stable and non-reactive compounds that bind withCYP1B1. The metabolism that resultsfrom this binding produces a metabolitethat induces apoptosis in the cancer cell.This is the basic discovery of the researchteam in Leicester, England.

Background Research and DevelopmentSince the work that I want to introducebuilds upon the earlier research on theanticancer relationship betweenSalvestrols and CYP1B1 it is important toprovide a brief history of thisdevelopment. There are two keyscientists behind this development: Prof.Gerry Potter and Prof. Dan Burke. Gerry isa medicinal chemist and cancer drug

designer. Dan is an emeritus professor ofpharmaceutical metabolism, and anexpert in cytochrome p450 enzymes andtoxicology.

Close to 20 years ago Gerry designed hisfirst prodrug. The London based Instituteof Cancer Research group that he waswith, at the time, were working onprostate cancer and had a great interestin CYP17. Gerry examined CYP17 in detail,worked out its binding site for metabolicaction and designed a prodrug thattargeted CYP17 to perform a terminalinhibition. The drug is called abirateroneacetate, currently known by the tradename ZYTIGA®. The clinical trials of thisprodrug have been exceptionallysuccessful. The company that has takenabiraterone through clinical trials, CougarBiotechnologies, just sold for $1billion toJohnson and Johnson on the strength ofthis drug. Given this drug’s performanceGerry has been named cancer researcherof the year by a UK prostate cancerfoundation.

Around the same time as Gerry wasdesigning abiraterone, Dan Burke beganhis investigation of a new p450 CYP1B1.He found it in soft tissue sarcomas. Helater found it in cancers of the breast andsubsequently found it a variety of cancersincluding cancers of the breast, colon,lung, oesophagus, skin, lymph node, brain,and testis with no detectable presence inhealthy tissue. Dan and his graduatestudents pioneered the research thatestablished CYP1B1 as a universal cancer

marker.

Dan and Gerry met some years afterthese two pieces of work. When Dan toldGerry of his exciting new enzyme Gerryimmediately drew upon his experiencedesigning abiraterone for CYP17 andstarted looking at how he could design adrug to target it. Within two weeks ofhearing of this enzyme Gerry designed aprodrug that was metabolised by CYP1B1to produce a metabolite that killed thecancer. Cell line results with this drugwere remarkable. The drug was perfectedover the next nine months, patented, soldoff and the long road to clinical trialsbegan.

During this time Gerry believed thatCYP1B1 was a natural rescue mechanismand if this hypothesis was true there hadto be analogues of his prodrug in food.Gerry led a search for these analogues,found quite a few of them and thesubsequent research programsurrounding salvestrols was born.Incidentally Gerry’s prodrug has anunbelievably high selectivity for cancercells – over the past few years naturalanalogues of this prodrug have beenfound that have vastly higher selectivitythan the prodrug that Gerry designed.

Need For New Clinical Tools

As the research developed conversationsabout the need for better clinical toolskept arising. The difficulty was twofold.

COVER STORY

COVER STORY

Current technology can only detectcancer once the cancer has achieved

between 108 and 109 cells (if you look atthe nail on your little finger, half that size

is between 108 and 109 cells – roughlythe size of a pea) – once cancer reaches

1012 cells (about a litre of cells) you’redead. By the time modern technology cantell you that you have this disease thedisease has silently grown through about75% of its life. Dan Burke wrote anexcellent article on this subject titled the

‘Silent Growth of Cancer and itsImplications for Nutritional Protection’.

The other side of the problem is that onceyou are told that you have this diseasethe clinical tools for monitoring diseaseprogression and treatment efficacy arepoor for most of the cancers.

Figure 1 has implications for people thatstart to think about cancer prevention.They, of course, assume that they are freeof the disease. They may consult aphysician and take advice on cancerprevention, but this advice is likely to alsoassume that they are free of the disease.However, they may lie anywhere on thatcurve below the level of detection. If theyare already up that curve preventivedoses are going to slow the rate ofgrowth but not keep the cancer frombreaking through into the detectablerange.

Figure 1 also has implications for peoplethat have journeyed through this diseaseto the point that their physician has toldthem that they are ‘all clear’. This

state of this disease. Basically we coulduse our understanding of this metabolicrelationship to report back to us on thedisease itself.

We took the decision to utilise thisknowhow and develop clinical tools forthe early detection of cancer, andtreatment efficacy. One thing that wehave learned so far on this project is thatit is a really good idea to have people onyour research team that don’t realise

‘that it can’t be done’.

In considering the problem we decidedthat we had one of two directions to take.The obvious first route was to develop amethod for detecting and measuring thepresence of CYP1B1 itself. Since CYP1B1is an intrinsic component of cancer cellsits detection and measurement wouldprovide a direct measure of the diseaseitself. The second and much less obviousapproach was to develop a method fordetecting and measuring the metabolicoutput of CYP1B1. If we could find astrong metabolic output of CYP1B1,detect and measure it we would haveanother direct measure of the output ofthis disease and by extension the diseaseitself. So we decided to pursue both –two universal tests for cancer.

The Proteomic Approach:In pursuing the detection andmeasurement of CYP1B1 itself we knewthat the job would be made much easierif we had antibody as this would help usto isolate what we were looking for fromall of the background material in humanblood, urine or tissue. We wanted anantibody to an amino acid string that was100% specific to CYP1B1, covering thewild form and the major polymorphs andnot found in any bacteria and one thatdidn’t have major cleavage sites runningthrough the middle of it. These criteriaruled out all of the antibodies that arecurrently available for CYP1B1. Weperformed an exhaustive search andidentified a set of peptides that met ourcriteria and embarked on raisingantibodies.

CYP1B1 is a very difficult enzyme to raiseantibodies for that have a strong affinityfor the peptide of interest becauseCYP1B1 is present in so many life forms inidentical form or near identical form to

Figure 1. The Silent Growth of Cancer.Reprinted with the kind permission of Prof. Dan Burke.

statement may simply mean that theirdisease is again below the level ofdetection. In the absence of ongoingtreatment and/or lifestyle change theircancer may break through into thedetectable range over the next few years.

All in all it amounts to a pretty miserablepicture.

It would be ideal to equip clinicians witha simple blood test that could be used toscreen for any of the cancers, with asensitivity that could pick up thepresence of the disease long before it has

reached 108 and 109 cells. Think abouthow much easier it would be for them toassist these people back to good health.It would also be ideal if a simple bloodtest could be used to monitor any of thecancers with a level of accuracy thatwould readily tell if a treatment isworking or not and whether a dose is highenough to be effective? A blood test thatis as applicable and accurate withpancreatic cancer as it is with breastcancer – a blood test that is as applicableand accurate with adrenal cancer as it iswith prostate cancer. Tools such as thesecould make life a lot easier for cliniciansand patients alike. Tools such as thesewould provide additional data that wouldhelp to advance our understanding ofhow best to prevent and treat the variouscancers and how best to keep them inremission.

Development of Clinical Tools.

The need for new clinical tools is obvious.One of the enormous implications of theprior work of Profs. Potter and Burke isthat it sets the stage for the realisation ofblood tests such as those that I justdescribed.

To embark on this work we looked atwhat we had to work with. We had greatexpertise on CYP enzymes, we had greatexpertise on secondary plant metabolitesand their metabolism by CYP enzymes.Specifically, we had CYP1B1, a universalcancer marker and salvestrols, naturalprodrugs, which in this context amountsto things to look for in the bodily fluids ofcancer sufferers. Given the salvestrol –CYP1B1 mechanism we knew that therewould be things that we could look forthat would tell us about the presence and

COVER STORYamidst a chorus of ‘I told you so’ until onemember of the team came up with thebright idea of starting with more blood!We increased the initial sample size anddetected and measured our nativepeptide.

ResultsThe naturally present CYP1B1 peptide wassuccessfully detected using antibody-affinity capture in both 20 µl and 200 µldigests of cancer patient plasma. Theamount of natural CYP1B1 in this samplecan be estimated to be ~200 amol/µl ofplasma. Upon achieving this result wehad an opportunity to replicate with 5additional samples.

With these results we performed somecalculations to determine where theseresults would fit into the growth curve ofthe ‘Silent Growth of Cancer’ andconcluded that these results wouldprovide detection of disease around 5.7years earlier than existing technology. Interms of the prognosis for lung cancer this5.7 year difference could be the differencebetween life and death.

that found in humans. However, we didmanage to raise an antibody to a specificCYP1B1 peptide and worked on affinityenhancement until we had somethinguseable.

Our first notion was to see if we coulddetect and measure CYP1B1 in humantumour samples. Seemed like a good ideaat the time – where else are we going tofind CYP1B1 in abundance?

We spent about a year working on samplepreparation methods and testing samplesusing some of the world’s mostsophisticated mass spectrometryequipment. We spiked the tissue matrixwith CYP1B1 from recombinant sourcesand managed to recover the recombinantmaterial but never managed to detect thenative CYP1B1. This caused us someconsiderable concern because the wisdomof the day dictated that if we couldn’tmanage to detect and measure CYP1B1 intumour samples, where it would beplentiful, we would never be able todetect and measure it in blood or urine.However, given that we were able todetect and measure the recombinant CYPfrom the tumour matrix we knew that wehad a sample preparation and extractionproblem – we were either not freeing theenzyme from the surrounding material orwe were destroying the enzyme with ourpreparation method.

In light of this we decided to abandon oursearch for CYP1B1 in tissue and focus ondetecting it in blood. This decision flew inthe face of conventional wisdom but ourthought was that if we were ever going tohave a viable diagnostic and monitoringtool it had to work on blood or urinesamples so if we were going to pound ourheads against a wall it might as well be thewall we needed to get to. This newstrategy isn’t really as crazy as it initiallysounds. When working with blood youdon’t need some of the sample prep stepsthat you would use with tissue becauseyou don’t have as much intact material todeal with – you are already working withfragments.

So we embarked on trying to find ourCYP1B1 peptide in blood and ended upwith the same results as we found fortissue! We spiked recombinant CYP1B1into blood and managed to recover it butwere unable to recover native CYP1B1

Lower levels of peptide were found inthese samples with the amount of naturalCYP1B1 ranging from 2 to 12.5 amol/ µl ofplasma. (MRM analysis was performed byinjecting 15 μl on an AB/MDS Sciex 4000triple quadrupole mass spectrometerequipped with a Nanoflow EksigentNanoLC-1Dplus HPLC, 5 x 0.3 mm C18Pepmap (5 μm particles) trap column, anda 75 μm x 150 mm Magic C18AQ (5 μmparticles, 100 Å pore size) analyticalcolumn using 30 min. analyses.)

Since achieving this early result we haverefined our methods and analysed plasmarepresented a small range of cancers. Wehave also analysed plasma from aproteomic standard to serve as ourbaseline. The proteomic standardrepresents pooled plasma from healthyvolunteers. We have detected CYP1B1 inthis proteomic standard at minute levelsthat can only represent the CYP1B1 foundin those tiny number of pre-cancerous orcancerous cells that are being killed off inhealthy individuals on a daily basis. Withplasma from cancer sufferers our bestresults to date have been found with lungcancer samples. When comparing CYP1B1levels to those in the proteomic standardthe levels in the lung cancer samples werevastly higher (between 92 and 6291 timesthat found in the standard).

Figure 2. Initial set of results -plasma levels of CYP1B1.

Figure 3. Level of detection ofCYP1B1 in lung cancer samples.

SummaryWe now have a sample preparationmethod and an antibody (an assay) that isable to directly detect and measure cancerthough detection of CYP1B1 in plasma.When we find our peptide in your bloodwith this assay you have cancer – thereare no false positives – you have cancer.

We now have a variety of methodenhancement experiments, stabilityexperiments, validation experiments andmethod transfer experiments to conductbut we know that CYP1B1 is present in theblood, we can find it and we can measureit.

What I really like about this approach isthat it will be simple and convenient forthe person getting tested. Simply put outyour arm for a sample collection like anyother blood test. What I also like aboutthis approach is that it is a direct detectionand measurement of the cancer itself andit is as applicable to pancreatic cancer asit is to breast cancer – it is applicable to allthe cancers. The other thing that I likeabout this test is that we are operating atan exceptionally high level of sensitivityand we have good reason to believe that

Sample Amount of CYP 1B1 (amol/-l ofplasma)

2ml tube labelled 22/04/2009 12.5

2ml tube labelled 23/04/2009 2

2ml tube labelled 24/04/2009 9.4

2ml tube labelled 25/04/2009 9.2

1.5ml brown tube non-labelled 4.9

COVER STORYanalysis the samples were prepared andBeta Glucoronidase was used to removethe sugar from the glycoside.

Following this we decided to take a lookand see if we could find a differencebetween healthy volunteers and thosewith advanced cancers. We administered1 gram of a specific salvestrol to eachindividual, waited 3 hours and drew theirblood. We also had each individual do a24 hour urine collection. As expected, withhealthy volunteers we found nometabolite – we simply recovered thesubstrate in blood and urine. Withdiseased volunteers the situation was verydifferent. We found a very clear spike onthe hplc where we predicted that themetabolite should come off the column.Some of these individuals had veryadvanced disease and with theseindividuals we found absolutely noaglycone and no glycoside – justmetabolite. When we analysed their urinewe also found no aglycone. The entiregram of substrate seemed to have beenused up. With other cancer patients wefound small amounts of aglycone alongwith large metabolite spikes. What thistells us is that the ratio of metabolite toaglycone may be of much greater clinicalvalue than the metabolite alone – time willtell. We performed these tests withindividuals representing a fairly broadarray of common cancers: breast, stomach,kidney, prostate, etc., and an array ofstages of cancer but skewed towards moreadvanced cancers. Metabolite spikes werefound for all as one would expect giventhat we are looking at the metabolicoutput of a universal cancer marker.

SummaryWe currently have a sample preparationmethod that allows us to detect theaglycone and the metabolite in blood orurine using hplc. We find clear separationsbetween the outputs obtained fromhealthy volunteers as compared todiseased volunteers. Like the proteomicapproach when we find this metabolite inyour blood you have cancer.

What I really like about this approach isthat it uses natural products as diagnostics.We are getting the metabolism of a naturalproduct to report on the presence andstate of disease. Another nice feature ofthis approach is that we can build thesignal by the amount of substrate that we

administer. An additional benefit of thisapproach is that it not only tells us thatCYP1B1 is present, that is that cancer ispresent, it can tell us that the enzyme isfunctioning fine.

There is also great scope for improving thisassay. We have been drawing blood at 3hours, the time of peak concentration forthe substrate. Conduct of a smallpharmacokinetic study to determine peakconcentration of the metabolite willbenefit this test greatly. Once we are ableto draw blood coincident with peakconcentration of the metabolite we willbe able to pick up the presence of cancermuch earlier. Like the proteomic test themetabolite test is universally applicable.

DiscussionA present we have two different assays fordetecting and measuring the presence andamount of cancer. Both operateindependent of any apriori notions abouttype of cancer that may be present. Thehuge strength of these approaches is thatthey can be used with all cancers – theyare two universal cancer tests that canultimately be used for diagnosis andmonitoring across all of the cancers. Thedownside of this is that we will need tovalidate both approaches on each andevery cancer.

Up untill now everyone on the team hashad their pet blood test – either themetabolite test or the proteomic test.However, from the onset there have beengood arguments for seeing both of theseapproaches through to completionbecause they provide differing set ofstrengths and weaknesses and when wecombine them we can potentially providemuch more clinical assistance than wecould with results from either one.

For example, let’s say that we have two36 year old females, very similar in familyhistory, medical history, etc., and bothhave a 2 cm cancerous lump in one of theirbreasts. Their physician decides to run themetabolite test. With one of the womena large metabolite spike is found with noaglycone and no glycoside. With the otherwoman we find a medium sized spike ofmetabolite and small spikes of aglyconeand glycoside. What is going on? With justthe metabolite test we might concludethat woman number 1 has fully

we can increase the level of sensitivityfrom here.

The Metabolite ApproachWe know the various substrates of CYP1B1,that is, we know what it metabolises andin particular we know a lot about thesalvestrols that it metabolises. So whathappens when we ingest salvestrols?

In our food salvestrols come in two forms:as a glycoside and as an aglycone – in foodabout 80% as glycosides and 20%aglycones – in supplements 100%aglycones. When we ingest the glycosidethe plant sugar is cleaved off and replacedwith a human sugar. When we ingest theaglycone a human sugar is attached. Thisof course assumes that everything isworking properly to perform this function.The new glycoside is then transported andupon reaching cancer cells the humansugar is cleaved off leaving the aglyconeat the cancer site. This step is performedby Beta Glucoronidase. The aglycone thenbinds with CYP1B1 and is metabolized. Themetabolite induces apoptosis spilling thecontents of the cancer cell, includingCYP1B1 peptides and metabolites into thesurrounding space.

What all this means for blood testdevelopment is that the interaction ofCYP1B1 and salvestrols provides us with avariety of measurable aspects of thisprocess that can provide us with insightsinto the presence of disease as certain ofthese aspects can only be present if thedisease is present and metabolism hastaken place.

We initially went through our list ofsalvestrols looking for metabolites thatwere abundantly produced throughCYP1B1 metabolism and not found in atypical diet. From a candidate list onemetabolite was chosen.

We looked to see if we could find theaglycone in blood and urine – initiallyusing predicted structures and then usingsynthesized standards we were able toreliably detect and measure the aglyconein both blood and urine. We thenperformed a pharmacokinetic study usinghealthy volunteers to determine whensalvestrols reach peak concentration inthe blood – three hours after ingestion.We identified the aglycone spike resultingfrom salvestrol using hplc. Prior to hplc

COVER STORY

disease around.

Along the way to reaching these goals weare able to add value to those volunteersthat contribute their blood throughadvising their physicians of the results.

functioning CYP1B1 that is making full useof the substrate while woman number 2may have competing substrates in herbody that are inhibiting the functioning ofCYP1B1 – for example she may have beenusing some household paints that containchemical anti-fungal agents or perhapsshe had the furnace duct work recentlycleaned and the cleaners used chemicalanti-fungal agents to retard any fungalbuild up or perhaps she takes daily walksalong the perimeter of a golf course thatuses a great deal of anti-fungal spraying.We may also conclude that womannumber 1 may have an additionalundetected tumour mass. If we now runthe proteomic test we can help todetermine what in fact is going on forthese two women. Let’s say that we runthe proteomic test and find a larger spikeof peptide for woman number 1 thanwoman number 2. This result would tellus that there may be no difference at allbetween the functioning of CYP1B1 forthese two women but rather confirm thatwoman number 1 has another,undetected tumour mass and this isaccounting for the higher results. Theattending clinician can then embark on asearch for the whereabouts of this secondtumour mass.

Current DirectionsWe want to keep pushing the limits ofdetection until we are picking this diseaseup at disease onset.

We want to be able to pick this disease upat points where simple dietary andlifestyle change can turn the diseasearound. That is one of our goals.

We also want to push the sensitivity ofthese tests to the point where we can tellwhether or not a treatment is workingwithin 24 or 48 hours after commencingtreatment. This will be exceptionallybeneficial to people pursuingconventional chemotherapy as it couldsave them from weeks of toxic exposure.

We also want to be able to utilise thesetests to individualise treatment plans forcancer sufferers.

We want to push the sensitivity of thesetests to the point that we can pick updisease recurrence at a point wheredietary and lifestyle change can turn the

Further reading:

Attard G, Belldegrun AS, de Bono JS(2005). Selective blockade of androgenicsteroid synthesis by novel lyaseinhibitors as a therapeutic strategy fortreating metastatic prostate cancer. BJUInt. 96 (9): 1241–6.

Attard G, Reid AHM, Yap TA, Raynaud F,Dowsett M, Settatree S, Barrett M,Parker C, Martins V, Folkerd E, Clark J,Cooper CS, Kaye SB, Dearnaley D, Lee G,de Bono JS (2008). Phase I Clinical Trial ofa Selective Inhibitor of CYP17,Abiraterone Acetate, Confirms ThatCastration-Resistant Prostate CancerCommonly Remains Hormone Driven.Journal of Clinical Oncology 26: 4563.

Burke, MD. (2009). The silent growth ofcancer and its implications for nutritionalprotection. British Naturopathic Journal,26:1, 15-18.

Burke, MD, & Potter, G (2006).Salvestrols ... Natural Plant and CancerAgents? British Naturopathic Journal,23:1,10-13.

Hoon LT, Butler PC, Burke DM, Potter GA,(2007) Salvestrols: A New Perspective inNutritional Research. Journal ofOrthomolecular Medicine, 22, 1: 85-93.

Li NC, & Wakeman M. (October 2009)High-performance liquidchromatography comparison of eightbeneficial secondary plant metabolites inthe flesh and peel or 15 varieties ofapples. The Pharmaceutical Journal,supplement Vol. 283, B40.

Li NC, & Wakeman M. (2009) High-performance liquid chromatographycomparison of eight beneficial secondaryplant metabolites in the flesh and peel or15 varieties of apples. Journal ofPharmacy and Pharmacology,supplement 1, A132.

McFadyen MCE, Melvin WT, Murray GI(2004) Cytochrome P450 enzymes: Noveloptions for cancer therapeutics.Molecular Cancer Therapeutics, 3: 363-371.http://mct.aacrjournals.org/cgi/content/full/3/3/363

McFadyen MCE, Melvin WT, Murray GI(2004) Cytochrome P450 CYP1B1 activityin renal cell carcinoma. British Journal ofCancer 91: 966‐ 971.http://www.nature.com/bjc/journal/v91/n5/abs/6602053a.html

McFadyen MCE, Cruickshank ME, MillerID, et al. (2001) Cytochrome P450CYP1B1 over-expression in primary andmetastatic ovarian cancer. British Journalof Cancer 85:242–6.http://www.nature.com/bjc/journal/v85/n2/abs/6691907a.html

McFadyen MCE, Breeman S, Payne S, etal. Immunohistochemical localization ofcytochrome P450 CYP1B1 in breastcancer with monoclonal antibodiesspecific for CYP1B1. Journal ofHistochemistry andCytochemistry,1999;47:1457–64.http://www.jhc.org/cgi/content/abstract/47/11/1457

McKay J, Melvin W, Ahsee A, Ewen S,Greenlee W, Marcus C, Burke M, MurrayG (1995) Expression Of Cytochrome-P450 CYP1B1 In Breast-Cancer FEBSLetters 374(2): 270-272.http://www.salvestrolscience.com/uploads/SAL/FEBS_Letters_1995_vol374p270-272.pdf

Murray GI, Melvin WT, Greenlee WF,Burke MD, (2001) Regulation, function,and tissue-specific expression ofcytochrome P450 CYP1B1. AnnualReview of Pharmacology and Toxicology.41: 297-316.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11264459&dopt=Abstract

Murray GI, Taylor MC, McFadyen MCE,McKay JA, Greenlee WF, Burke MD,Melvin WT (1997) Tumor specificexpression of cytochrome P450 CYP 1B1.Cancer Research, 57: 3026-3031.http://cancerres.aacrjournals.org/cgi/content/abstract/57/14/3026

COVER STORY

Murray GI, McKay JA, Weaver RJ, et al:(1993) Cytochrome P450 expression is acommon molecular event in soft tissuesarcomas. Journal of Pathology, 171:49–52,http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8229456&dopt=Abstract

Potter GA, Burke DM (2006) Salvestrols –Natural Products with Tumour SelectiveActivity. Journal of OrthomolecularMedicine, 21, 1: 34-36.

Potter GA (2002) The role of CYP 1B1 asa tumour suppressor enzyme. BritishJournal of Cancer, 86 (Suppl 1), S12, 2002.

Potter GA, Patterson LH, Wanogho E etal (2002) The cancer preventative agentresveratrol is converted to theanticancer agent piceatonnal by thecytochrome P450 enzyme CYP 1B1.British Journal of Cancer, 86: 774-778.http://www.salvestrol.ca/BJCreprint.pdf

Schaefer BA. April 2012. Salvestrols,Nature’s Defence Against Cancer. ClinicalIntelligence Corp.Schaefer BA, Potter GA, Burke DM,

Wood R, (2012). Cancer and Related Case

Studies

Involving Salvestrol and CYP1B1. Journalof Orthomolecular Medicine, 27, 3: 131-138.

Schaefer BA, Dooner C, Burke DM, PotterGA, (2010) Nutrition and Cancer: FurtherCase Studies Involving Salvestrol. Journalof Orthomolecular Medicine, 25, 1: 17-23.

Schaefer BA, Hoon LT, Burke DM, PotterGA, (2007) Nutrition and Cancer:Salvestrol Case Studies. Journal ofOrthomolecular Medicine, 22, 4: 177-182.

Ware WR, (2009) Nutrition and thePrevention and Treatment of Cancer:Association of Cytochrome P450 CYP1B1With the Role of Fruit and Fruit Extracts.Integrative Cancer Therapies, 8, 1: 22-28.http://ict.sagepub.com/cgi/content/abstract/8/1/22

Ware WR, (2009) P450 CYP1B1 mediatedfluorescent tumor markers: A potentiallyuseful approach for photodynamic

therapy, diagnosis and establishingsurgical margins. Medical Hypotheses, 72:67-70. http://www.medical-hypotheses.com/article/S0306-9877(08)00428-3/abstract

About the author:

The author was educated in Victoria, B.C., Canada and Oxford, England, obtained a B.Sc.,and M.Sc., degree from the University of Victoria and a Doctor of Philosophy (D.Phil.)degree from Oxford University in England (Wolfson College). After these studies werecompleted he chose to return to Canada. After two years as a research fellow in Ottawahe returned to Victoria where he currently lives with his wife and his two children. Afondness for England continues and he returns to England on a regular basis. He haspublished and lectured on a broad array of topics including psychometrics, patternrecognition, visual perception, knowledge acquisition, artificial intelligence, laboratorymedicine and cancer research.

The cover story of our first issue is an in depth explanation of CYP1B1, If you have missedit then it is available on our website as a download. The following is an extract from it:

Cytochrome P450 enzymes (abbreviated as CYPs and pronounced 'sips') belong to asuperfamily of proteins which contain iron at their core and often referred to as'haemoproteins'. These CYPs are a large and diverse group of enzymes that use a varietyof large and small molecules as substrates in enzymatic reactions. Being enzymes they arenatural biological enabling devices which change structures of chemicals in the bodywithout themselves becoming changed. CYP enzymes have a long history and originated3.5 billion years ago. Their name is a composite one derived from having a cellularlocation(cyto), a spectrophotometric characteristic (chrome) and a unique opticalabsorbance peak at or near 450nm.

CYP enzymes are ubiquitous and found in all life forms from animals, plants and fungi. Morethan 11,500 distinct Cytochrome P450 enzymes are known to exist. In humans there are57 different CYP proteins wich are all bound to membranes of the endoplasmic reticulumor to the inner membrane of the mitochondria and as such have distinct cell-specific,tissue-specific and developmental-stage-specific characteristics. The majority of CYPs areto be found in the liver, however, individual forms have been identified in extra-hepatictissues such as lung, kidney and intestines.

Right: CYP1B1

CLINICAL CORRESPONDENCECase Study:

Low grade papillary urothelial carcinoma,kidney cancer and pancreatic cancer.B.A. Schaefer, D.Phil.(Oxon)

A 61-year-old male presented to hisdoctor with macroscopic haematuria,swollen ankles and fungal infections onhis nails (Onychomycosis with concurrentParonychia). He was a non-smoker with ahistory of good health outside ofdeveloping septicaemia following a kneeoperation eighteen years earlier. Hisbleeding persisted for two days and hisdoctor, although suspecting an infectionand prescribing an antibiotic, referredhim for an ultrasound. Two weeks lateran ultrasound revealed an abnormalthickening near the left vesico-urethraljunction. He was referred for flexiblecystoscopy.After a period of seven months flexiblecystoscopy revealed a small papillarytumour near the bladder neck close tothe left vesico-urethral junction. Anenlarged prostate was also noted. He wasreferred for surgery.Two months later a transurethralresection of bladder tumour (TURBT) wasperformed. Surgery revealed a 2cmpapillary growth at the bladder neck andoverlying left ureteric orifice. No obviousdisease was found in the ureter. A singledose of epirubicin was installed.Paracetamol (Panadol tablets) 500mg,twice daily along with tramadolhydrochloride, 50mg capsules, 1-2 daily,were given for pain relief but declined.Sodium citrotartrate (Ural) granules, 1sachet daily, was also prescribed. Duringsurgery it was discovered that the tumouralso involved part of the urethra andlesions were also found on a kidney. Thesurgeon felt that the cancer had beenremoved from the bladder and urethrabut was concerned about the kidneylesions and unsure about whether thebladder cancer would return. Theswelling in his ankles was quickly resolvedpost-surgery.The histology report indicated that agrey/brown tumour, 20mm x 17mm inaggregate was received post-surgicallyrevealing a low grade papillary urothelialcarcinoma with no invasion present. Hewas referred to an oncologist. Theoncologist indicated that the tumour hadbeen a fast growing tumour that wasquite rare. It was larger than they had

anticipated. Flexible cystoscopy andmagnetic resonance imaging (MRI) werescheduled as part of a post-surgery,follow-up.Seven months following surgery flexiblecystoscopy indicated a tumour on thepancreas. An MRI was performed sixweeks later revealing a 40mm tumour onthe pancreas. The oncologist informedthe patient that he had a life expectancybetween 10 and 20 weeks. The patientbecame overcome with fear as he wasvery familiar with the prognosis forpancreatic cancer due to his priorinvolvement with pancreatic cancerresearch fundraising.Coincidently a former colleague had beenin touch and upon hearing of the cancerdiagnosis suggested that he supplementhis diet with Salvestrols. On threeseparate occasions his colleague madethis recommendation and his suggestionwas declined. Two weeks following thediagnosis of pancreatic cancer he agreedand commenced daily Salvestrolsupplementation comprising twoSalvestrol Platinum capsules for a totaldaily intake of 4000 Salvestrol points. Thiswas added to his daily regimen of 500mgof vitamin C, a fish oil capsule and anevening primrose capsule. No otherchanges to diet and lifestyle were madeat this time although there was a modestmove towards more organically grownfoods. Daily exercise had always formedpart of his routine.Twelve weeks following his MRIconfirmation of pancreatic cancer andeight weeks following the onset ofSalvestrol supplementation flexiblecystoscopy indicated slight shrinkage ofthe pancreatic tumour. Follow up MRIconfirmed the shrinkage and revealedconcurrent shrinkage of the kidneytumour. Salvestrol supplementation wasmaintained along with his usual dailysupplements. No further changes weremade to diet or lifestyle.Four months later and six months afterstarting Salvestrol supplementation afurther flexible cystoscopy found noevidence of pancreatic or kidney cancer.Given this surprising result an MRI wasscheduled. The MRI confirmed the findingof the flexible cystoscopy. Due to thehighly unusual finding a second MRI wasperformed which also confirmed thefindings of the flexible cystoscopy. Anastonished oncologist told him that hewas cancer free and that the kidney had

healed very well. A follow up flexiblecystoscopy and MRI were scheduled in ayear’s time.During the intervening period he hasdecreased his daily Salvestrolsupplementation to one SalvestrolPlatinum capsule per day for a daily totalof 2000 Salvestrol points. He hascommenced a daily, one hour walk andmoved his diet to include organicallygrown foods when ever practical to do so.He is feeling great and enjoying goodhealth. Twelve months following hiscancer free status he reports that he feelsten years younger and his recurrentproblems with nail fungus(Onychomycosis with concurrentParonychia) have also been resolved.Salvestrol cream was applied for thispurpose.As the date of his one year follow up forflexible cystoscopy and MRI approached,Salvestrol supplementation wasincreased to two capsules comprising atotal daily intake of 4000 Salvestrol pointsand then three capsules for a daily intakeof 6,000 Salvestrol points. The one yearflexible cystoscopy found no evidence ofbladder cancer, pancreatic cancer orkidney cancer. His cancer free status wasconfirmed and it was felt that there wasno need for an MRI. He will be testedagain in a year’s time.

IJOPThe International Journal Of Phytotherapy

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Diabetes: A modern disease

Insulin is a hormone in the body that regulates blood sugar.Over time, raised blood sugar (hyperglycaemia) can lead toserious damage of the body’s systems, particularly theblood vessels and nerves.

In the long term, diabetes can damage the heart, bloodvessels, eyes, kidneys, and nerves. It increases the risk ofheart disease and stroke, blindness and death.

There are many people who have diabetes and remainundiagnosed until complications arise.

Diabetes and its complications have a significant economicimpact on individuals, families, health systems and countries.

A new diagnostic test for diabetes

Scientists have recently discovered a new and inexpensiveway of testing for diabetes.

The test is based on a known diagnostic marker, calledglycated hemoglobin (HbA1C).

In the life of a red blood cell, glucose molecules react withhemoglobin to form glycosolated hemoglobin. Inindividuals with high blood sugar due to poorly controlleddiabetes, these molecules are more prevalent.

The test is an accurate, low cost, robust method ofdiagnosing diabetes. Furthermore, the test can detectthese molecules in a spot of dried blood, making thismethod suitable for mass screening a population.

Today 346 million people worldwide suffer from diabetes, a chronic diseasewhen the body does not produce enough insulin, or cannot effectively usethe insulin it makes. With 1 in every 20 people suffering from diabetespopulation screening and cost effective monitoring becomes essential.

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Dried blood spot extraction

Modification with the reagent

Blocking excess of the reagent

Trypsin digest

MALDI mass spectrometry

Data analysis

Report of the glycation values

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Early detection is key

One of the biggest problems facing physicians in thetreatment of cancer is early detection. Although thereare some screening tests,cervical smears, mammograms.All of which are imperfect tests as they are dependenton the skills of the pathologist looking at the samplemany people are left to discover they have cancer whenthey experience unusual symptoms or discover a lump.Unfortunately, at this point the cancer can already bequite far advanced and may have even spread to otherorgans, making treatment much less effective.

A new screening method that will save lives

A unique enzyme called CYP1B1 that is produced bycancer cells. The importance of this enzyme has lead to arevolutionary new blood test that has been developed todetect any cancer at very early stages of its developmentif cancer is found early enough, simple changes to dietand lifestyle could be enough to prevent the progressionof the disease. This is a huge step forward in cancerdiagnosis and treatment. It is estimated that one third ofall cancers could be cured, using current conventionalmethods, if detected early and treated appropriately. Thiswould mean that this new cancer test could save millionsof lives worldwide every year.

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Our technology will identify the significant presence of cancer cells without the need for any apriori idea of where in the body the cancer is located. In so doing it provides a relativelyinexpensive first stage indicator as to which individuals should go for more expensive scanningand biopsy cancer tests, and it also enables clinicians to assess the progression of disease andprovide an indication of the success or otherwise of any selected management programme.

Significant investment and directed research has already taken place to establish thefoundations for this new patented technology and as a result we have identified a number ofdifferent approaches which have the potential to be executed using current commonly availableanalytical techniques used in commercial laboratories.

Our two lead researchers have a wealth of experience in cancer discovery behind them andthese advances represent a new approach to the conundrum of how to assess and evaluate thepresence of cancer in an individual. Our approach uses simple analytical assessments of bloodor urine specimens and in the future, possibly breath, without the need for invasive methods,biopsies or highly expensive and complex in-vivo monitoring devices.

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