Novel phosphonate containing ligands for optimised targeted radiotherapy of neoplastic bone disease : using animal models and scintigraphy

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Novel Phosphonate containing Ligands for

optimised targeted Radiotherapy of Neoplastic

Bone Disease: using Animal Models and

Scintigraphy

Irene C. DormehI

H.Sc (Phpics, Ma~hemnfics), M.Sc. (Physics),

D.,%

@uclear Physics)

Thesis submitted in hlfilment of the requirements for the degree:

Pharmacology

At the:

North-West University

Potchefstroom Campus

Promotor: Prof. Douglas W. Oliver North-West University

Potchefsrroom Campus

C+promoter: Dr. Werner K.A. Louw Ilniversity of Pretoria

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Novel Phosphonate containing Ligands for

optimised targeted Radiotherapy of Neoplastic

Bone Disease: using Animal Models and

Scintigraphy

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Summary

This thesis wvers research on radiotherapy of bone cancer using bone seeking radiophmaceuticals i v. applied. Such research generally has been confined lo the treatment of bone pain in people with metastalk bone cancer but on occasion also for lreating cases of osteosarwma It is typically used when there are multiple metastatic lesions in the skeleton, which makes local and focal t~eaiment impractical and systemic treatment the alternative. The treatment is tumour specific since the radiopharmaceulical targets the area of increased mineral turnover. This a l l o m selective uptake and prolonged radiopharmaceu tical retention in these areas.

An ideal radiopharmaceulical for the treatment of neoplastic bone disease would be a radiolabelled compound. w h c h would predominantly accumulate in the bone lesion with limited access to normal bone and other organs.

Criteria governing the selection o f the radionuclide are parlicle range, physical half-life,

gamma yield (for scinligraphic monitoring), chemistry and type of ligand. The most commonly used radionuclides

ar

present are phosphorus-32, strontium-89, lin-1 17m, samarium- 153, and rhenium- 186/ 188.

Currently the available bone seeking agents which are phosphonaie containing ligands lend to localize throughout the skeleton. This thesis focuses on melhylenediphosphonic acid (MDP),

1- hydroxy-ethylenediphosphonic acid (HEDP), and the novel agent polyethyleneirnine functionalised with methylene phosphonate groups (PEI-MP) from this laboratory, as isell

as

the octa-anion ethylenediaminetetramethylphosphonate (EDTMP). MDP and HEDP are bisphosplionates, EDTMP a muhidentate arninophosphonate. In targeted therapy EDTMP is used in combination with I5%m and might be binding. to hydroqapatite crystals in a different manner than E D P .

'"R~-EDP is used for palliation of bone pain to metastatic bone cancer. Complications known with '"s~-EDTMP and '=R~-HEDP t~eatment are myelotosicity and in some cases a transient increase in bone pain folloning treatment (flare response).

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In contrast to this '"~e-HEDP appears ro fulfil the criteria of a good radiotherapeulic agent as it has a stronger b-emission than I5%m and l g 6 ~ e and a shorter physical and biological half- life which reduces myelotosicity.

The aim of [his research is to optimise and investigate novel bisphosphonate containing ligands for targeted radiotherapy of neoplastic bone disease using rodent and primate animal models and scintigaphy.

The successful outcome of hese studies requires that the normal primale (Popio ursinus) wilh its in vivo bisphosphonate behaviour for purposes of scintigraphic monitoring, (using T c - MDP), be validated as a usefid model to interpret esperimental results and esl~apolale to man as was done successfully in h e fracture healing experiments discussed here.

The pharmacokinetics of 1 5 3 ~ m - ~ ~ ~ ~ ~ . the well known herapeutic radiopharmaceutical used for palliation in painful human skeletal metastases, was determined in normal primates by scintigraphic monitoring and compared with available human data in order once more to establish suficient similarity of in vivo behaviour and thus direct the meaningful wntinualion of these biodymarnic investigations.

A novel approach for ligand optimisation involving neoplastic tissue's abnormal blood supply (increased permeability) and lack of lymphatics (EPR effect) was investigated, whereby radiolabelled macromolecules accumulated selectively al the target site. The synthesis of the macromolecule polyethyleneiminomethyl phosphonic acid (PEI-MP), and its labelling with '"Tc, as well rls quality control have been described in detail. Macromolecular sizes ranged

from 3 to 300 kDa and the label w"bTc was selected to senre as tracer for the scintigraphic biodistribution studies performed in normal adult male primates. The results allowed the identification of a suitable size-fraction, i.e. 10-30 kDa of PEI-MP, as a bone-seeking ligand. Molecular sizing of h e polymeric PEI-MP can drastically alter its pharrnacokinelic properties.

To esploit hese encouraging results, of organ sparing and speedy urine excretion a study of bone tumour accumulation of '*'Vc-PEI-MP was done in five dogs with spontaneous occuring appendicular osteosarcomas. Mean tumour: background uptake of 4: 1 was obtained with the molecular size fraction 10-30 kDa To fulfil a therapeutic role h e most suitable

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Summary

ligand would have lo form a slable and appropriate complex wilh one or more of the therapeutic radionuclides e.g.

"%in,

lg6Re and 1 L 7 m ~ n ( l ~ ) .

For informed selection of such a radionuclide which would be successfully complesed with the ligand PEI-MP for targeted delivery to osteosarcoma/metastatic bone lumours, metal ion speciation in blood plasma was used to predict fie in vivo behaviour of the potential bone- seeking therapeuric radiopharmaceuticals. The blood plasma model ECCLES used here,

included PEI-MP as ligand, and predicted good in vivo behaviour of " 7 m ~ n ( 1 1 ) - ~ ~ ~ - ~ ~ . but nor so when complexed with 15%m and 16%Io. Labelling of PEI-MP with "7m~n(11) and also

wilh IB6Re was subsequently successllly achieved,

It is known that changing the radionuclide label of a particular ligand might change the

resultant biodistribution. Therefore the biodistribution of variously molecular sized 1 L 7 m ~ n ( ~ ~ ) - polyethyleneitninomethyl phosphonale complexes were investigated in the normal primate

model to establish their potential as selective therapeutic bone agents. The in vivo stability of l I 7 m _{Sn-PEI-MP, and reduced accumulation in the kidneys and normal bone observed from} these biodislribuiion studies prompted the investigation of its potential to exploit the EPR effect due lo its macromolecular nature, where bone malignancies are present. The tumour uptake of " 7 m ~ n - ~ E I - ~ ~ in different types of canine osteosarcoma i n d u d into nude mice,

was therefore studied.

The osteosarcoma model followed from subcutaneous injection of canine osteosarcoma cells with high lung metastatic capacity (HMPOS) in some mice, and without this capacity (POS) in another group. The former yielded faster growing non ossified lumours compared to slower growing tumours from POS cells, with ossified tissue, both observed histologically. The high accumulation of " 7 m ~ n - ~ ~ ~ - ~ ~ in the bladder \ d l is of great concern and a focus of continuing research.

There is however no doubt that the ligand PEI-MP eshibits promising characteristics for targeted delivev of therapeutic radionuclides to neoplastic bone lesions and essentially spares vital organs and normal bone.

The normal primate f h e r m o r e turned out to be a userul model from which to interpret scintigaphic results related to in vivo bisphosphonate behaviour, d s o for scintigaphic

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Summary

monitoring o f the phmacohnetics and biodislribution o f bone therapeulic radiopharmaceuticals,

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Opsomming

-

Nuwe Fosfonaatbevattende Ligande vir die

optimaal teikengerigte Radio tera pie van Neoplastiese

Beenletsels deur gebruik

van Dier Modelle en Sintigrafie

Hierdie proefskrif handel oor navorsing in radioterapeutiese behandeling van beenkanker deur gebruik te maak van beensoekende radiof'aseutika wat binneaars toegedien word. Oor die algemeen was sodanige navorsing dusver beperk tot die behandeling van beenpyn in persone met metastatiese beenkanker, maar by geleentheid ook vir die bebandeling van ge\.alle van osteosarkoom Dit word tipies gebruik waar daar veehuldige melastatiese letsels in die skelet aanwesig is, wat plaaslike en fokale behandeling onprahies maak en sistemiese behandeling die alternatief is. Die behandeling is tumorspesifiek aangesien die radiofarmaseulika die area van vermeerderde rnineraalomsetting as teiken het. Dit maak voorsiening vir selektiewe opname en verlengde radiofarmaseutiese retensie

in

daardie ateas.

'n Ideale radiofarmaseutikum vir die behandeling van neoplastiese beenletsels sou 'n radioaktief gemerkqe verbinding wees wat hoofsaaklik

in

die beenletsels akkumuleer met beperkte opname dew normale been en ander organe.

Kriteria wat die keuse van die radionuklied bepaal, sluit in die reihwydte van die partikels, fisiese halfleeflyd, gamma-lewering (vir sintigrafiese monitering), chemie en die tipe ligand Die radiondiliede wat tans die meeste gebruik word, is fosfor-32, strontium-89, tin-1 17m, samarium- 153 en renium-1861188.

Die beensoekende middels tans beskikbaar, naamlik figande wat fosfonate bevat, neig om deur die totale skelet te versprei. Hierdie proefsbi f konsen~reer op metileendi fosfoonsuur (MDP). 1-hidroksie-etileendifosfoonsuur (HEDP) en 'n nuwe middel poli-etileenimien gefunksionaliseer met metileenfosfonaatgroepe (PEi-MP) (uit hierdie studie), mook die okta- anioon etileendiarnientetrametielfosfonaat (EDTMP). MDP en HEDP is bisfosfonate, EDTMP is 'n multidentaataminofosfonaa~. By teikengerige terapie word EDTMP saam

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met lS3sm gebruik en sou moontlik bind a m hidroksiapatiet kristalle o p 'n ander y s e as

HEDP.

'"R~HEDP word gebruik om beenpyn by merastaliese beenkanker te verlig. Bekende komplikasies, a voorkom by 1 5 3 ~ m en - ~' B 6 ~ e - ~ ~ ~ - b e h a n d e l i n g , ~ ~ ~ ~ is mielotoksisiteit en in sommige gevalle, 'n beenpyn van verbygaande aard wal nAbehandeling aanwesig is (flare response). ' 8 8 ~ e - ~ ~ ~ ~ voldoen in 'n groter mate aan die krileria van 'n goeie radiolerapeutiese agent, in die lig van die sterker bela-uilstraling teenoor die van ""m en '"Re. asook 'n korter fisiese en biologiese halfleertyd het, met gevolglike verminderde mielotoksisiteit.

Die doel met hierdie navorsing is om nuwe Iigande \\.at bisfosfonaal beva te optimaliseer en hul m n a n d i n g te bestudeer by teikengerigte radioterapie van neoplastiese beenletsels deur sebrui k te maak vam sinti y a fie en knaagdier- asook primaatmodelte.

Die suksesvolle uitkoms van hierdie studie sou vereis dat die in vivo bisfosfonaatgedrag in die normde gesonde primaal/Kaapse bobbejaan (Papio ursinus) vir doeleindes van sintigrafiese monitering. (dew gebruik te maak van '*c-MDP), bevestig moet word, om sodanig as 'n geskikte model te dien vir inlerpretering van eksperimentele resultale en vir ekstrapolering na die mens, In hierdie doel is suksesvol geslaag soos aangetoon deur

fraktuurgenesingseksperimente.

As gevolg van abnormale bloedtoevoer (verhoogde pernieabiliteit) na en gebrek aan limfvate in neoplastiese weefsel, sou radioahief gemerkte mahomolekules (bisfosfonate) selektief akkurnuleer in die letsels.

Hierdie vorm dus die basis vir 'n nuwe benadering van ligand optimalisering. Die sintese van die makromolekuul poli&tileeniminometiel fosfoonsuur (PEI-MP) en die merking daarvan met v c , sowel as h-waliteiiskontrole word breedvoerig bes kryc Makromolekulbe yoottes wal wissel van 3 tot 300 kDa en v c is gekies as spoorder ('[racer') vir die ~ i n t i ~ e s e biodinamika studies uitgevoer op normale volwasse manlike primate. Die resuliate maak die identifisering van fraksies met geskikte groottes, naamlik 10-30 kDa van PEI-MP, as 'n beensoekende ligand moondik. Die grootte van die mahromolekules van die polimeriese PEI- MP kan die farmakokinetiese eienskappe drasties verander.

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Opsomming

In die lig van belowende resultate van orgaanbeskerming en snelle uitskeiding in urien is 'n 99

ondersoek gedoen, t.0.v. beenlumor akkumulasie van "Tc-PEI-MP, in vyf honde met spontane appendih11Cre osteosarkomas. Gemiddelde tumor tot agtergrond-opnames van 4: 1 is v e r b met molekulGre rraksie-grootte van 10-30 kDa Om die terapeutiese doel le bereik, moet die mees geskikle ligand 'n stabiele en toepaslike verbinding vorm met een o r meer van die terapeutiese radionukliedes, bpoorbeeld 15'~m, lS6Re en "7"~n (11).

Vir die ingeligte keuse \.an so 'n radionuklied, wat s~lksesvol bind met die ligand PEI-MP, om die osteosarkoom / metastatiese beentumors te leiken, was metaalioon spesiering in bloedplasma gebruik om die in vivo gedrag van die potensieel beensoekende terapeutiese radiofarmaseutika te voorspel. Die bloedplasma-model ECCLES wat h e r gebruik is bevat PEI-MP as ligand en het goeie in vivo respons van " 7 m ~ n ( 1 1 ) - ~ ~ 1 - ~ ~ aangedui \vat nie die geval was met I5%m en l M ~ o komplekse nie.

Merking van PEI-MP met 1'7m~n(11) en met '%Re was d m a suksesvol uitgevoer. Dit is bekend dat die verandering van die merker (radionuklied) van 'n spesifieke ligand die biodistribusie moontlik kan verander. In die lig hiervan is die biodis~ribusie van verskeie molekukre grooltes van H7%n(11)-polieti~eeniminometiel~os~onaat verbindings ondersoek by die normale primaalmodel om die geskihlheid te bepaal as selekliewe terapeutiese beenrniddels. Die in vivo stabiliteit van 1 ' 7 m ~ n - ~ ~ 1 - ~ ~ en die veminderde akkumulasie in die niere en normale been soos uit hierdie biodistribusie studies waargeneem was, het as

aansporing gedien om die potensiele toepassing te bestudeer van die EPR-effek (Verhoogde permabiliteit en relensie effek) by been turnore ag.1,. die rnakromoleh~l&e eienskappe van die polimeer by die Lurnore. In die lig van die laasgenoemde was die tumor-opname

1 l7n1

ondersoek van Sn-PEI-MP in verskillende soorie honde-osteosarkomas, in muise getnduseer.

Die osteosarkoommodel volg vanuit die subkutane inspuiling van die selle van honde- osleosarkoom met hoe longmetastaliese kapasiteit (HMPOS) in sekere muise en sonder hierdie kayasiieit (POS) in die ander g o e p . Eersgenoemde het snellergroeiende nie- ossifiserende tumore gelewer teenoor die stadigergoeiende tumore van POS-selle met histologies bevestigde ossifiserende nreefsel. Die hoe voorkoms v a n " 7 n 1 ~ n - ~ ~ 1 - ~ ~ in die blaaswand, is kommerwekkend en gevolglik ook 'n fokus van voorigesette navorsing.

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Opsommiag

Hierdie navorsing het duidelik aangetoon dat die PEI-MP-ligand belowende eienskappe toon vir teikengerigte lewering van terapeutiese radionukliedes aan neoplastiese beenletsels, sonder om lewensbelangrike organe en normale been le benadeel.

Die normale primaat blyk ook 'n geskikte proefdier te wees om sintigrafiese resultate varl bisrosfonaatgedrag le inlerpreleer, w o k vir sintigrafiese monitering van die farmakokinetika en biodist-ribusie van beenterapeutiese radiofarmaseutika

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Acknowledgements

I would like to sincerely thank rhe following persons for their respeclive contributions to the work presented here:

-

Pro[ Douglas Oliver, as promoter and valued collaboralor, for his assistance and guidance towards compiling the data and information eminating From this estensive research into a volume which could widely serve to direct future similar investigations. He was an enthusiastic collaborator and participant in an even wider field of investigation for more than

I5 years during which I could benefit from his considerable knowledge in his field of espertise and share and enjoy with him his dedication to science in general. It is also solely through his vigorous administrative eITorts that this thesis at all c c m e to be. For especially this, Douglas, words of thanks fail me.

-

Dr. Werner Louw for his painstaking scrutiny of dl the facts and figures which appear in this manuscript. As an important partner in these studies, he was not just the backroom boy in the radiochemistr_v laboratory, but rather the man behind new and innovative ideas. It was each time during many years an esciting esperience to test yet another compound in vivo, and watch the intellectual reasoning become reality.

-

Dr. Rowan Milner for kindly sharing his esperience and expertise, especially in the treatment of canine osteosarcoma and developing o f osteosarcoma cell lines for the rodent sludies.

-

Dr. Jan Rijn Zeevaarl for modelling metal ion specialion in bloodplasnln and for initiating and direcling the invesligations with " 7 m ~ n ( ~ ~ ) obtained from the Netherlands.

-

Dr. Frank Schneeweiss for enthusiastic international networking.

-

Prof. Filomena Botelho for making facilities and stalT available to continue with the rodent studies at IBILI, University of Coimbra It is on her shoulders that the responsibilities for the future research will rest.

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Acknowledgments

-

Dr. Ralf Clauss for some clinical informarion

-

Me. Elmare Kilian for expert and dedicated technical assistance including caring and handling of the experimental animds, for many years.

-

Prof. Antoine van Gelder for kindly taking care of the logstics which provided the infrastructure which for many years sustained the research and whose efforts also greatly supported the completion of this thesis.

-

Me. Marianna Bergh for putting together so deAly all the scribbled pages o f paper which became this book. That was the most dificult part. Marianna you are a star!

-

Prof. Piet de Kock for translations.

Arranging the daia and informalion from a near lifetime of research naturally brought with it

moments of reflection. It n;as From these moments that 1 acuiely recognized with humble gratitude and awe the hand that steered destiny for this, and much more, to fall into place, not only to produce a PhD thesis, but redly to shape a life.

In this light I w.ould like to dedicate this thesis to my mother who for dl these years shared the woes, and savored the successes with me, and to Johann, my pillar of strength, who, when

dl else seemed to fail. made sanity prevail,

My thanks and appreciation to the following Institutions:

-

University of Pretoria under the auspices of which the work was done

-

University of North West for recent support

-

University of Coimbra for support with the rodent studies

-

Research Centre Julich through support of Dr. Schneeweiss

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Acknowledgemenis

-

NRF and the Bundesministerium Deulschland for bilateral travel funding between Germany and South Africa

-

Alesander van Humboldt Foundation, Bonn-Bad Godesberg, for sponsoring periods of research on many occasions.

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in

the Normal Primate 14.3 Osleosarcoma Bearing Nude Mice and Normal Wislar Rats

14.3.1 Comparison behveen Pharmacokinelics of Y c - a n d "7"~n(11)-~~1- MP

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Table of Contents

14.3.2 Work in Progress

14.4 References

Chapter 15: Conclusion

Appendix 1.: Glossary of Abbreviations and terms

Appendix 2: Reprints

Appendix 3: Lette~s of Consent

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Table

of Figures

Figure 2-1 Calcitrm in hlrman bone. Sewn@ percent of /he calcirrm .sirlts conlaining aburrt 25, 000

mmol nre in crysmllized table state. Thirly percent uf the calcirrm salts contnining nborrt 100 mmol are in nmorphot~.~ exchnngeable state.

Figure 2-2 The RANK-RANKL system in osteolytic bone metnstnvis.

Figure 2-3 Model of osteoblastic bone metavtnsis cnriscd by prostate cmcer.

Figure 2-4 Phosphonates mil phosphate.^: their uses, molectrIar weight and chemical structure. Figure 2-5 14ision mo!vbdenrrm generator (Peltek F) interior view.

Figure 2-6

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"7mSn-P~~-h4P q~rlirJ, control-example, (b) representative chromntograms of imptrrc Ph"~r.-lnbelledprod~~cts.

Figure 2 7 (a) Gamma cnmern ns a rrnit and (Fr) the cmera hmd.

Figure 2 8 (a) Collimator viewjrom the top and (b) Jiom the sidc. (r.) ,5?hematic dingram (crms section) oj'n parallel hole collimator: only pnrallel entering r q s will pass to the crystal.

Figure 2-9 Schematic rcpresentntion of scintillntion detection processes in the crystnl and phutomrrltiplier tube.

Figure 2-10 Operatur/compnter inte$ace throtrgh terminal and displqv processors. a keyboard and nrrsor4rnckL.r ball control.

Figure 2-1 1 finmple o J ' n scintigraphic image of n rat with @pic01 regions of interest (IZOIs) CZeJined.

Figure 2 12 Examples of dparnic and 96 oorgnrlistribrrtion gryhs, as obtnincdfiom corrnt raes temporally ncquired

Figure 2 1 3 The normd human skeleton, anterior and posterior views Figure 2-14 Non lrnion bone with rndiotracer lingering around fracture site Figure 2-1 5 O.vteosnrcoma, very marked rrptnh with well vi.srralised mnrgins Figure 2 1 6 Metastatic bone trrmonrs, anterior and poslerior views

Figure 2-1 7 .Schemntic presmtation of .selective hone trrmnru 1ocali.r.ntion

Figure 2- 18 Organ and tumorrr uptoke o f99"Tc-- PH-MP, depending on its mncromolecular size Figure 2-19 Prepnred cmimnI Jbr scintigrphy. The size of the animnl ennhles meaning/id datn extrnyolation to man.

Figure 2 2 0 (a) A lwo-comynrtmmt Jystcm with only two transfir cuejlicients and (b) An isolated two-compnrtment .yvstem with no tranfirs to and/rom the outside.

Figure 4-1 Tlw Mennen clamp-un plate in position on n cadax ' r j' orcarm. Figure 4-2 Normal fi~rearm andproJiIe; elbow and wrist arem clendv indicated Figure 4-3 Three dqvs post-operative trmrma visible un the proJile of the right ulna.

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Table of Figures

Figure 4 4 (a) I:'nrly henling (in/lnmmaoty) stage 3 week7 po,st-operarive. The prt,jile is of the right rrlna The implnnt is a Mennen plate and (b) A three w e d posr-uperatiw srtrdy with a srandnrd 4 hole Milller plnle. The projile is of the right radius.

Figure 4-5 (a) A six week post-operntive sludy wilh a Mennen plate. Profile uflelf ulna and (b) Stric& oJ'n 4-hole Miiller plnte 6 week7 posl operative. I'rojile ofthe right rudirrs

Figure 4-6 A typicnl late stirdy with n Milller plate distingrrishing between " Jinctrrre" nnd "screw" activi)y,

Figure 4-7 L/N vs rimc cuntcs. (a) Menncn implant. (b) Shmman implant. (c) Mijller implant. Figure 5-1 Typicnl scintib~ams uf bnboons indcating skelelal lrcalisation of (4 *TC-MDP, (b)

I J J

Sm-EDTMP ( I I I MRq). and (c) r m ~ o - ~ D ~ (I 85 MBq) 4 h posrinjecticm.

Figure 5-2 Time nctivity curves (corm~.s~~ixcl) Jor the bluodpcrol (x), liver (o), kidhry (o), nnd backgrotmd (+) obtinedfi.um dynnmic srudies (0 to 30 min pstityection) with I I IMRq ' S J ~ m - EDTMP (4 and I85 MBq r 6 6 ~ o - ~ D ~ ~ l j (b), re.~pctive!v.

Figure 5-3 Percent bone ~q?tnkc (region of interest-hip) oJ'l85 MRq * T C - M ~ ~ P , 185 MBq lmHo- EDTMP, and I I I MRq " J ~ m - ~ ~ ~ ~ l J 4 to 7 hours pos~ injection. SD ranges ore intiicnted, Figure 5-4 Coml)nrtmenml annlysis shuwingfit~ed nrrves for [he bloodpr~l (x), wine (o), bune (m), kidney (o), nnd remninder of the bor3, ( r ) of " 3 , % - ~ D ~ M P (4 and r M ~ o - ~ l > ~ M P (b). Figure 6-1 Mean (n - 6) time acntivi)y cun3es Jiom the 2-h @namic study ofthe cardiac blood

pool (a), kidney (b) nnd liver (c) ajkr 0-week (m), 6 week (01, and 12-week ( A ) npp1icntion.r of ' S 3 ~ m - ~ D ~ M P with low concentrntion oJ' IXYIiW"I) Mean (n - 6) blood clearance (d), and cumulnlive urine vnh!es (e) o f r S J ~ m - ~ ~ T M P (low concentration) tahn uwr 5 h nJler 0-week (m).

6 week ( m ) , and 12-week ( A ) application

Figure 6-2 Histugrnm of mean percentage bone rrptakt. o j " S 3 S m - ~ f l ~ (low concentrnriun) obtained from 2 h, 4 h and 5 h stnric scintigraph.~ aJ/er @week, 6 week and I week npplicorions

Figure 6-3 Mmn (n - 6) lime aclivify crrrves from the 2 h dynamic sttrdy of carrliclc. blood pool

('n), kidney (61, and liver (c) aJer the &week (m), 6 week (m), and 12-week ( A ) applications qf 1 J 3 . ~ m - ~ l l ~ ~ wirh h;gh mncenmation EDlfMP. Mean blwd clearnnce ([I) and armrrlntiw wine valtles (el o f r J 3 S m - ~ ~ 7 * ~ P (high cononcLenrrarion taken over 5 h nJrr &week (B), 6 week (0). and

1 '-week ( A ) applicntions.

Figure 6-4 Histogram of mean percentnge bone t,ptnke of 1 J 3 ~ m - ~ ~ l ~ (high concentration) ohtninerl from 2 h. 3 h. 4 h nnd 5 h static scinligraphy a f i r 0-week, 6 we& nnd 1Bwcek npplicationv.

Figure 7-1 PercAenrnge uf retnined but& activiy Jor drfirent -TC-PEI-M~' Jinctions in the primae liver. - Frncliun 3-10, l -- Fraction 1 &30. A - Fraction 30-50, V - Fraction 50-1 00,

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Table of Figures

Figure 7-2 Menn percentage of re1ninc.d boo) nclivity for dflerenl *li.-~Ii~-~P/i-acrions in Ihe primale kiclney. - Fraclion 3-1 0, - Froctron 10-30, A - Fmction 30-50, 7 = Fraclion 50-

100 kDn

Figure 7-3 Normdizetl blood clearance Jbr dflrenr *Tc-PEI-MP. - Fraction 3-10, =

fiaclion 10-30. A -. Fraction 30-50, 7 - fiachbn 50- 100,

+

Ikclion l W 3 0 0 kIh.

Figure 8-1 Uptake of QR*~c-l~olqmin (10-30 m a ) into various organs of lhe Dnlnmalion as a Jrrnclinn uflime (I hr dynamic sttc& and up 10 3 hr slnlic sltro)).

Figure 8-2 Dorsoven~ml *TC-PE~-MP (10&300 k h ; I85 Mq) bone scan of n llog

(Dnlmnlion) with ostcowrcoma uf lhe righl scnptda (95% scapln involvemenl).

Figure 9-1 The ligand EI3'IiUP and polyrner repenling units discussed in lhis paper.

Figure 9-2 1ij;perimentol (pints) and modelled (lines) deprotonntion

( L ) )

cwves for Cn(1l) -

cumplex.nriun by Ihe repeating rutif of'l'li'l-MP. 7'he dashed line is the n crrrve and represents the prolonamm . s ~ n ~ e of'rhe lignnd in the absence of lhe melal ion. The Jive sepnrale lilralions crre represenled by (0) 0.000X5Z mot dm" C(ll), 0. &I869 mol dm-' repding mil of PI: *I-MP and

0,00997 mot dm3 HCl; (0) 0.000852 rnol h-' Ca(lI), 0.001 74 m d dmJ repeating mil of PEi-MP nnd 0.00997 mol dm" HC'I and (A) 0.00M52 mol dm-' Ca(II), 0.00261 mol dm repenling rutil uf' ITI-MP nnd 0.00989 mol dm-'HCl; (0) 0.00128 mot dm" Ca(II), 0.001 00 rnol repealing rrnil of PEI-MP nnd 0.01 02 rnol dm" HCI and (

9

0.001 70 mot dm" Ca(ll), 0,001 00 ma1 dm" repealing rrnil of PH-MP nnd 0.001 01 mot dm" HCI; wrws 0.0500 mot dm" NaOH in 0.01 0 mot

cbn" NnCl. All solutrom were nt 259. nnd 0.15 mot dm" NaCl or 0. I5 mot dm" ~olal ionrc strength.

Figure 9-3 ,Ypecres dislribrtlion cirrves for Cn(1I) complexaliun by PEI-MP d 25°C: and 0.15 mot dm-' 0,s c*nlcdaled fiom the formnlion comtanfs in Table 9-2 Concenrrrions rrsed were

0.000852 mu1 dmJ Ca(II) and 0.001 74 mot dm" repealing itnil of PEl-MP.

Figure 9-4 F~perimenlnl (uuinls) and modelled (lines) deprolonn~ion

(3)

cunw for &(,I) -

complexntion by the repealing rutit (IJ'PFJ-MP. The dashed line is the n irrrve nnd represcnrs the prolonation .sIale of the lignnd in the absence of the metnl ion. TheJive separate litrations are represenred by (o) 0.0001 00 mot dm" Znfl), 0.001 00 mot dm-' repenting rmit

14'

PL .I-MP nnd 0,00990 rnol dm'' HTI; (0) 0 001 00 mu1 dm" Zn(lI), 0.00201 mol h" repeating unit of YU-MP

and 0.0@97 mot dm" HC'I and (A) U.OOOI00 mot dm" %n@l). 0.00301 mot h -3 repealing mil of' PEI-MP and 0.009 71 mol drn"H~1; (0) 0.00201 mol dm--' Zn(I1). 0.001 00 mot ch" repenling rutil of PEl-MP and 0.0102 mol dm" HCl and (

9

0.00150 rnol dm3 Zn(ll), 0.00100 mol dn"

repealing rutit ofP13-MP and 0.001 01 mol dm" HCI; versus 0.0500 mot dmm-' NaOH in 0.01 0 rnol

h-' NnCI, All soltrlions were a1 25°C and 0.15 mu1 dm--' NnC'I or 0.15 mot dmJ Iulnl ionic slrengrh.

(24)

Chapter 1 -

Introduction

1.1 Background

The field of research reported on in !his thesis covers procedures of radiotherapy of bone cancer using bone seeking radiopharmaceuticals i.v. applied. To a large extent such research generally has been confined to the l~eatment of bone pain in people with metaslatic bone cancer but on occasion also

in

cases of osteosarcoma (Goeckeler er al.. 1987; Holmes 1992;

Bruland et al., 1996; Serafini, 2001), It is typically used when there are multiple melastatic lesions in the skeleton. which makes local and focal trdment impractical and systemic treatment an attractive alternative (Lewington, 1996). The treatment is tumour specific since the radiopbarmaceutical targets the area of increased mineral turnover. This allows for selective uptake and prolonged radiopharmaceutical retenlion in these areas (Goeckeler er al.,

1987). 11 also sugests thal treatment would be more e f k t i v e in predominantly sclerotic lesions associaled with brisk os~eoblastic reaction lhan in destructive lesions. Since the treament is localized to the active lesions in certain parts of the skeleton, tosicity is reduced minimising damage to healthy tissue. However this is no1 always the case as myelolosicity occurs with high doses of radiopharmaceuticals (Laltimer er ol., 1C990; Bayouth er ol., 1994;

Milner er 01.. 1998; Franzius er al., 2002; Anderson er al,, 2002).

An ideal radiopharmaceutical for b e treatment of neoplastic and inflammatory (benign) bone diseases would be a radiolabelled compound, which would predominantly accumulate in the bone lesions with limited access to normal bone and other organs. (Bouchet el al., 2000)

Criteria governing the selection of the radionuclide are parlicle range, physical haw-life, gamma yield (scintigraphic monitoring), chemistry and type of ligand. The most commonly used radionuclides al present are listed in Table 1-1 (Athns, 1998; Liepe er ol., 2000; Body &

Mmcini, 2002).

Currently the available bone seeking agents which are phosphonate containing tisands tend to localize throughout the skeleton. This thesis focuses on methylenediphosphonic acid (MDP), 1- hydrosy-ethylenediphosphonic acid (HEDP), and a novel agent polyethyleneimine

(25)

Chapter 1 - lnlraluclion

fimctionalised with methylene phosphonale groups (PEI-MP), as we!l as the octa-anion ethylenediaminetetrmethylenephosphonafe (EDTMP). MDP and HEDP are bisphosphonates, EDTMP a multidentateaminophosphonale which tends to form a more stable chelate with fewer structural forms than HEDP (Lin, 1996). In targeted therapy EDTMP is used in combination with '"Sm and might be binding to hydrosy-apatite crystals in a different way than HEDP, In a review by Serafini (2001) the effect of EDTMP on bone neoplasia was mentioned to require furlher investigation as it was thought that the ligand in itself may effect

186

bone neoplasia Re-HEDP is used for palliation of bone pain to metastatic bone cancer. Complications known with ' 5 3 ~ mand I s 6 R e - H E ~ ~ - ~ ~ ~ ~lreatment are myelotosicity and ~ in some cases a transient increase in bone pain following treatment (flare response) (Farhanghi el al.. 1992; Brenner er al., 200 1 ; Liepe el al., 2005). In contrast lo this '%e- HEDP appears lo fulfil the crileria of a good radiotherapeutic agent as it has stronger

P-

emission than "'~m and ' 8 6 ~ e and a shorler physical and biological half-life which reduces myeloloxici ty (Palmedo el al., 2000; Li er al., 2001 ; Liepe el al., 2003; Palmedo el a/. ,2003).

Research currently focusses on optirnisaiion of phosphonate containing ligands carrying therapeutic radionuclides. These are designed to reach a target area and be retained there as a radiopharmaceutical to achieve optimal therapeutic efficacy. Some of the ligands used in radioisotope therapy are themselves pharmacologically active agents and may even contribute to the function of the radiopharmaceutical (Klenner el al., 1990; Body ef al., 1998; Diel, 2000; Body & Mancini, 2002). HEDP is k n o w as etidronate and is used therapeutically in osteoporosis in humans where it inhibits bone resorption (Manolqas, 2000).

An alternalive and novel approach for ligand optimisation is to use neoplastic tissues' abnormal blood supply leading to increased permeability, as well as lack of lymphatics, which will lead to setectively accumulated radiolabelled macromolecules at the target site (Seymour, 1992). 'Ibis enhanced permeability and retention effects (EPR) ( M d a er al., 2001) form the basis of the unique study presented here using various molecular sizes of the radiolabelled

macromolecule y c polyethyleneimjnomethylphosphonate (witten as w m ~ ~ SO - as ~ ~ ~ - ~ ~ ) to increase selectivity by the ligand, of bone seeking radiophmaceuticals. This effect is

applicable only to macromolecules and lipidic particles. not to low-molecular-weight compounds. the calegory to which most drugs in use today belong (Maeda el ol., 2001). Low molecular weight compounds are distributed fieely by Wusion to various tissues and organs; the compounds move against the concentralion gradient until finally an equilibrium results.

(26)

Chapter 1 - lnlrduction

Their concentration in tumours cannot be higher than in blood plasma, nor can they be retained at high concentrations in tumows for a significant time period because of rapid excretion (washout) into the bloodstream The plasma concentration also diminishes rapidly as a result of efficient renal clearance via the urine (Maeda ef ai.. 2001). In contrast.

macromolecules and polymeric drugs are retained in lumour tissue at a much hlgher concentration than in plasma (Maeda el a!., 2001). There is thus a peat difference between

low- and high-molecular-weight compounds in their intratumour accumulation. This phenomenon, the Enhnced Permeobilily ond Refenfion (EPR) elfect, is now recognized as a general characteristic of viable and rapidly growing solid tumows. Another general characteristic is the structural deficiencies of tumow blood vessels. which also cause enhanced leakiness (Maeda e f ni., 200 1).

Table 1-1 : Physical Properties of Therapeutic Radionuclides

I

Nuclide I

(

Conversion

I

Half-life (days)

I

Samarium- 1 53

1

1.9

1

3.7

/

0.8 Rangein

I

Beta-emission Rhenium- 186 3.8 Gamma-

1

Abundance

1

tissue (mm) Em, (MeV) emission

/

(Oh.)

-

-2.5

Molecular siting of polymeric PEI-MP can drastically alter the pharmacokinetic properties (Dormehl e f nl., 200 I), which can be esploited to suit dmerent applications and targeting of specific organs, tissues and pathological affected a r m . The localisdon of PEI-MP is also essentially in the surface areas of bone tumours (Milner, personal communjcation). In order to obtain an optimal molecular size, which would largely exclude damage to normal bone and other organs, the phannacokinetics, i.e. biodistribution of various '39"Tc-PEI-MP size fractions could be invest igaed using various groups of experimental baboons (Papio ursinrds). Once an

electrons

(27)

Chapter I - lntrduction

optimal molecular size is identified it could be used in rodent experiments eventually using osteosarcoma bearing nude mice, The murine model is a familiar approach and useful for development of tumour seeking ligands to enhance targeted radiotherapy (Pool el nl., 1988).

Informarion concerning the in vivo behaviour of drug formulalions following administration to human subjects can be obtained from radionuclide imaging techniques in investigative procedures (Siegel el ol., 1999). In general the radiolabelling can be achieved with g m m a - emitting radionuclides e.6. '*c as tracer and monitored using a gamma camera The

153 186.188

therapeutic isotopes Sm, Re can in low activity imaging dosages sen1e as its own tracer with the relevant accompanying ligands.

The nonhuman primate has always served as an extremely suitable animal model in drug development and analysis, even in the blery complex field of neurology, where remarkable similarities to human anatomy and physiology exist (Connolly, 1950; Le Gros Clark, 1960; Fridman & Popovq 1988; Fridman & Popova, 1988; Louw el al., 1991). The advantage of this model together with regular nuclear medical equipmen! for scintigraphy lies in its size which will allow flexibility in extrapolating raw data from images, as well

as

of evaluated data to man. Dosimetry is an esample of where the primate model becomes very useful.

Before embarking on bone seeking drug development however the baboon and its bisphosphonate bone biodynamics had to be studied, i.e, in fracture healing esperiments, where the pattern of bone healing could be scinliyaphicalb, monitored. The applicability of the results from 'h"Tc-MDP images thus obtained, to humans would encourage subsequent studies to establish the pharmacokinetics of ' S % m - ~ ~ ~ ~ ~ in the normal primale. Since such data obtained from gamma camera scintiyaphic monitoring are also available for humans, good agreement would validate the use of the primate model for future novel drug development.

An established primate model, would permit novel phosphonate ligands to be studied. The biodistribution of variously sized macromolecules of radiolabelled polyethyleneiminomethyl phosphonic acid

as

a selective bone seeker for therapy could thus be studied in the normal primate model, bearing in mind sparing the bone marrow, kidneys and liver from escessive radialion exposure.

(28)

Chapter 1 - Inlrduction

Meld ion speciation i.n blood plasma could be used to predict the in vivo behaviour of !he potential bone-seeking therapeutic radiopharmaceuticals. The object here is to construct a blood plasma model which includes PEI-MP (May el al., 1977; Janis & Wagener, 1995). This would enable informed selection of the radionuclide for delivery to osteosarcoma and metaslalic bone tumours by PEI-MP. This gives an indication of the abilily of the radiopharmaceutical to sunjve competition for the radionuclide by other blood plasma

166 ligands. Therapeutic fbpariicle emitting radionuclides under consideration are ""m, Ho,

89 lsb'188

s

r, Re, as well as the Auger electron emitter "'"~n.

To evaluate the therapeutic potential of suitably labelled PEI-MP the biokinetics of e.g. the 10-30 kDa fraction (in diagnostic activities of radionuclide) could conceivably be studied

in

canine osteosarcoma bearing nude mice (Balb C) (Kadosawa el al., 1994). Osteosarcoma is the most common bone tumour in man and accounts for 20% of all bone malignancies. It

furthenno re resembles canine os teosarcoma m his tological appearance and biological behaviour (Owen, 1976: Brodey, 1979; Knapp & Waters, 1997; Macewen, 1990; Hahn et al.. 1994). This accounts for the approach taken here to model bone neoplasia from nalurally occuring canine osteosarcoma in rodents (Parodi. 1982; Pelfrene, 1985; Pool er al., 1988). It is also the closest to modelling human metastatic bone cancer, although the process involved

in the production of osteol>lic and osteogenic metastases differs from the abnom~alities, largely of chromosomal origin, in osteosarwma cells. Diagnosis of metastatic disease is similar for osteosarcoma and include radiographs, CT, scintigraphy and MRI (Forrest el al.,

1992: Leibman er al., 2001: Davis er ol., 2002; Wallack er al., 2002). The therapy for both is commonly directed at palhion of bone pain, and the drugs generally used consist of non- steroidal anti-inflammalory drugs. narcotic analgesics and bisphosphonales also wdh radionuclides as in this study (Straw er al., 1990; Milner at al., 1998; Ramirez el al., 1999; Tomlin er al., 2000).

Sythetic polymers provide a broad technology platform for applications in the pharmaceutical sciences (Alesander, 2001). A variety of compounds can be coupled directly or with a selection of appropriate linkers to an amino group containing the ligand PEI-MP to form many different possibIe polymer-drug conjugates. In the area of drug delivery, especially to tumours and idlammalory areas, PEI-MP could have a unique and novet role to play also without a radionuclide label.

(29)

Chapter 1 - Intrduction

1.2 Aim

and Objectives

In view of the discussion above the aim of this research was to investigate novel bisphosphonate containing ligands for targeted radiotherapy of neoplastic bone disease using rodent and primate animal models and scintiyaphy. The success of the outcome sets very specific requirements for the sequence of investigation as is described in the section on experimental design (Chapter 3).

This required sequence directs the specific objectives of the study as follows:

(i) (i i) (iii) (iv)

( ~ 1

(11 i) (uii) (v ii I)

To validate the primate model for its bisphosphonate bone melabolism in fracture healing where the pattern o r bone healing was scintigraphically monitored and compared to human images.

T o establish the pharmacokinetics and biodistribution of the known therapeutic radiopharmaceutical agent "%n~-EDTMP scintigraphically in primates, compare it

lo human data and thus validate the primate model for this experimental approach. T o design a novel ligand with optimised tumour localisation potential sparing the other vilal organs by making use o r the EPR principle.

T o confirm the in vivo characteristics o r this novel ligand (PEI-MP) through biodistribution studies in the normal primale model as established above, using scintigaphy with '*c labelling of the ligand.

T o perform metal ion speciation in blood plasma (ECCLES model) to predict the in vivo behaviour of this novel potential bone-seeking therapeutic radiopharmaceutical, labelled with various therapeutic IS-emitting radionuclides in order to allow informed selection of the radionuclide delivered to a bone lesion. T o develop labelling techniques of the ligand ~ f i l h I B 6 ~ e , and/or l17"Sn, in order to obtain the required therapeutic radiopharmaceuticd.

T o investigale the labelled ligand '""~n-PEI-MP scint igraphicall y for suitable biokinetic and biodistribution properties of tumour targeting initially in the normal primate model.

To evaluate the potential therapeutic properties of "'"Sn-PEI-MP, through biokinetic biodistribution studies in canine osteosarcoma bearine nude mice. and

(30)

Chapter I - ln&cduclion

10 calculate the radiopharmaceutical uptake by various tumours. This would indicate the therapeutic potential of the labelled ligand.

1.3 Study Design

The procedures used in this investigation include the highly specialized techniques of isotope production in a nuclear reactor (Safari-I, NECSA, Pelindaba), and the associated radiochemistry of isotope separation, and preparalion to deliver the radionuclide to be used in the radiopharmacy laboratory for labelling the ligand, which had been designed through various synthetic chemistq considerations.

In vivo studies of the labelled ligand using the non-human primate model and rodents (Wistar rats and Balh C mice) will be performed with scintigraphic procedures (gamma camera), and organ counting in a well counter (rodents). These biodistribution studies will take place with the animals under controlled anaesthesia Theoretical mathematical modelling will provide information on appropriate radioouctide selection (ECCLES), dosimetry (MIRD) and compartmental distribution of the r a d i o p h m a c e u t i d ,

These studies will be performed after approval by the Ethics Committee of the University of Pretoriq according to the pidelines of the National Code for Animal use in Research, Education and Testing of Drugs and Related Substances in Souih Africa. These guidelines are in line with international standards.

1.4 Presentation of Thesis

The reader is reminded that this thesis is presented in the formal, whereby the methods, results and discussioos relating to the various studies were incorporated into the ten papers (chapters 4-13. see 1-5). Attention is drawn to the f a t that the ten papers have already been published internationally. Also note that the references to chapters 4-13 are presented as in rhe original publications.

(31)

Chapter 1 - lnlrduclion

1.5 Publications

Chapter 4: A Technique to Evaluate Bone Healing in Non-Human Primates Using Sequential

9'

yc- ethylene

diphosphonate Scintigraphy. Nucl. Med.2 1 : 105-1 09. (1 982)

Chapter 5: Evaluation of Samarium-153 and Holium-166-EDTMP in the Normal Baboon Model. Nucl. M d . Bid. 23:935-940. (1 996)

Chapter 6: Uptake of Ehylenediamine Tetramethylene Phosphonic Acid in Normal Bone after Multiple Applications: A non-human primate study. Armeim.- fi,rscfr/ L h g Rcs. 48(4):408-4 14. (19%)

Chapter 7: Biodistribution and Pharmacokinetics of Variously Sized Molecular

Radiolabelled Polyethyleneiminomethyl Phosphonic Acid as a Selective Bone Seeker for Therapy in the Normal Primate Model. Arzneim.- ForscWDrug. Res. 54: 258-263. (2001)

Chapter 8: Optimalisation of Radiolabelled Polymin-MP of Different Molecular Sizes

as

selective Bone Seeker for Therapy in Animal Models. Physics M d i a 17: 53-55. (2000)

Chapter 9: Metal ion Speciation

in

Blood Plasma Incorporating the Water-soluble Polymer, Polyethyleneimine Functionalised with Methylenephosphonate Groups, in Therapeutic Radiopharmaceuticds. Ihdiochim. Acm. 90: 237-246, (2002)

Chapter 10: " 7 m ~ n and I g 6 ~ e radiolabelling of polyethyleneiminomethyl phosphonic acid (PEI-MP), a potential selective therapeutic bone tumour seeker. Nucl. Med. Chmm. 23: 1223- 1224 (2002), presented at the 10" Biennual Congress of the South African Society of Nuclear Medicine, Stellenbosch, 4-7 Dec 2002,

Chapter 11: Biodistribution and phmacokinetics of Variously Molecular Sized 1 ' 7 m ~ n ( ~ ~ ) - Polyethyleneimonomethyl Phosphonate Complexes in the Normal Primate Model as Potential Selective Therapeutic Bone Agent. Arzneim - 1;urscWDrug res. 54, No. 6,340-347. (2004)

(32)

Cbapler 1 - hllruduclion

Chapter 12: "7"'~n and Re Radiolabelling of Polyetyleneiminomelhyl Phosphonic Acid (PEI-MP), followed

b s

In Vivo Targeting of Induced Osteosarcoma in Nude Mice. Nucl. Mcd./Nukl. Med. 42: 156-157 (2004), presented at Radio Isotopes in Clinical Medicine and

Research, 26Ib Internalional Symposium, Bad Gastein, Auslria, 13- I6 Jan 2004.

Chapter 13: Comparison of Tumour Uplake in Different Types of Osleosarcorna Mice as

Studied with the Potential Bone-seeking Rndiopharmaceulical, " 7 n S n ( l l [ ) - ~ ~ ~ - ~ ~ . World J. Nucl. Med. 3: 237 (2W4). Inlernalional Congress of Radiopharmacy and Radiochemistrj-, G d m k .

(33)

Chapter 1 - Introduction

1.6 References

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ANDERSON, P.M., WISEMAN, C.A., DJSPENZIERI, A., ARNDT, C.A., HARTMANN, L.C., SMITHSON,W.A. er 01. 2002. High-dose samarium-1 53 ethylene diamine tetramelhylene phosphonate: low toxicity of skeletal irradiation in patients with osteosarcoma and bone metastases. J (Jin Oncol20(1): 189-1 96.

A m S , H.L. 1998. Ovenriew of Nuclides for Bone Pain Palliation. Appl Radiur hor 49(4):277-283.

BAYOUTH, J.E., MACEY, DJ., KASJ, L.P., FOSSELLA, F.V. 1994. Dosimetry and tosicily of samarium-153-EDTMP administered for bone pain due to skeletal metastases. .I Ntrcl. Med. 35(1):63-69.

BODY, JJ., BARTL, R., BURCKHARDT, P., DELMAS, P.D., DTEL, I.J., FLEISCH, H. el 01. 1998. Current use of bisphosphonates in oncology. International Bone and Cancer Study Group. J Clin Oncol 16( 1 2):3890-38i99.

BODY, JJ., MANCJNI, 1. 2002, Bisphosphonates for cancer patients: why, how, and when'? Suppurr Chre Cancer 10(5):399-407.

BOUCHET, L,C., B O L C H , W,E., CODDU, S.M., HOWELL, R.W., RAO, D.V. 2000. Considerations in the selection of radiopharmaceuticals for palliation of bone pain from metastat-ic osseous lesions. .A Nucl. Med.4 1 (4):682-687.

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BRODEY, R.S. 1979. The use of naturally occurring cancer in domeslic animals for research into human cancer: general considerations and a review of canine skeletal osteosarcoma Yale J B i d Med 52(4):345-361.

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Chapter l

-

introduction

BRULAND, O.S., SKRETTJNG, A., AAS, M. 1996. Targeted radiotherapy of osteosarcoma using 153 Sm-EDTMP. A promising approach. Acfn Clnco/35:38 1-384.

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DAVIS, G.J., KAPATKIN, A.S., CRAIG, L.E., HEJNS, G.S., WORTMAN, J.A. 2002. Comparison of radiography, computed tomography, and magnetic resonance imaging Tor evaluation of appendicular osteosarcoma in dogs. ./Am Vet Med Assoc 220(8): 1 171 -1 176.

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DORMEHL, I. C, L O W ,W. K A., SCHIYEEWEISS, F. H. A., MILNER, R, SCHMITT, G., CROFT, S. A. 1998;Uptake of ethylenediarnine lelra-melhylene phosphonic acid in normal bone aner multiple applications

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A primale study. Armelm.- I;orschhi D n ~ g Hcsmrch 4 8(4):408-4 1 4.

DORMEHL, LC., LOUW, WKA., MILNER, R.J., KILIAN, E., SCHIYEEWEISS, F.H.A. 200 1. Biodislribulion and pharmacokinelics of variously sized molecular radiolabelled polyethyleneiminome~hyl phosphonic acid as a selective bone seeker for lherapy in the normal primate model. Arzneim. -Forsch.!dmg Res. 5 1 : 258-263.

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1458.

FORREST, L.J., DODGE, RK, PACE, R.L., HEIDNER, G.L., MCENTEE, M.C., NOVOTNEY, C.A. e/ a/. 1992. Relalionship between quantitative lurnor scintigraphy and time lo metastasis in dogs with osteosarwma. J Nucl Med 33(8): 1542-1 547.

FRANZIUS, C., SCHUCK, A, BIELACK, S.S. 2002. High-dose samarium- 153 elhy lene diamine teharnelhy lene phosphonale: low tosici t y of skeletal irradiation in patients nith osteosarwma and bone metastases. J Ciin Oncol20(7): 1 953- 1 954.

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Chapter 1

-

lnlrduction

FRIDMAN, E.P., POPOVA, V.N. 1988. Species of genus Pupio (C'ercopirheciu'ue) as

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Chapter 1

-

Inlroduction

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LIEPE, K., RUNGE, R., KOTZERKE, J, 2005. The benefit of bone-seeking radiopharmaceuticals in the treatment of metastatic bone pain. J. Cancer Rex C'lin. Oncol.

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Hop 58(30-31): 1731-1735.

Novel phosphonate containing ligands for optimised targeted radiotherapy of neoplastic bone disease : using animal models and scintigraphy

Novel Phosphonate containing Ligands for

optimised targeted Radiotherapy of Neoplastic

Bone Disease: using Animal Models and

Scintigraphy

Irene C. DormehI

D.,%

Thesis submitted in hlfilment of the requirements for the degree:

Pharmacology

At the:

North-West University

Potchefstroom Campus

Novel Phosphonate containing Ligands for

optimised targeted Radiotherapy of Neoplastic

Bone Disease: using Animal Models and

Scintigraphy

Summary

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Opsomming

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Nuwe Fosfonaatbevattende Ligande vir die

optimaal teikengerigte Radio tera pie van Neoplastiese

Beenletsels deur gebruik

van Dier Modelle en Sintigrafie

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Acknowledgements

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Table of Contents

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Chapter 1

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Introduction

1.1

Background

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