Uptake and localization of 99mtechnetium-methylene-diphosphonate ...

IHIS-mf—9924

Uptake and localization of 99m technetium-methylene-diphosphonate in bone

99mTc(Sn)-MDP

99mic-MDP

»»•• T.J.F. Savelkoul

Uptake and localization of technetium-methylene-diphosphonate in bone

99m

99m

(opname en lokalisatie van technetium-methyleen-diphosphonaat in bot)

(met een samenvatting in het Nederlands)

Proefschrift Ter verkrijging van de graad van doctor in de Geneeskunde aan de Rijksuniversiteit te Utrecht, op gezag van de Rector Magnificus prof.dr. O.J. de Jong, volgens besluit van het college van decanen in het openbaar te verdedigen op dinsdag 12 juni 1984 des namiddags te 2.30 uur

door Theodorus Johannes Franciscus SAVELKOUL geboren op 30 december 1947 te Grevenbicht (L)

PROMOTOR:

prof.dr. S.A. Duursma

CO-PROMOTOR: dr. S.J. Oldenburg

Voor de uitgave van het proefschrift werd financiële steun verleend door het 3A-Fonds van de Kliniek voor Inwendige Geneeskunde van het Academisch Ziekenhuis Utrecht.

STELLINGEN

'.

Via electrolytische reductie van "^Technetium-pertechnetaat in aanwezigheid van Methyleen-diphosphonaat is het mogelijk een ^^mTc-MDP complex te vormen.

2.

Bij gebruik van ^^mTc(.Sn)-HT)P vindt de opname van het radionuclide in bot plaats na hydrolyse van het complex.

3.

De uitscheiding van "mTc(Sn)-MDP en "mTc-MDP in de urine vindt plaats in de vorm van het intacte complex.

H.

Bij een toename van de a c t i v i t e i t van osteoblasten zal de opname van ""mTc-MDP in bot ter plaatse eveneens toenemen.

5.

Alle onderzoek bij patiënten, anders dan anamnese lichamelijk onderzoek, i s aanvullend onderzoek.

6.

Onafhankelijk of er bij een milieuincident een bevolkingsonderzoek wordt overwogen, dient er op individuele basis een mogelijkheid te worden gecreëerd voor medisch onderzoek van mensen met gezondheidsklachten, die worden toegeschreven aan blootstelling aan de verontreinigende stoffen.

7.

Het bepalen van de verhouding van stabiele lood isotopen in het bloed van kinderen met een verhoogde loodbelasting, alsmede in monsters afkomstig van mogelijke bronnen, kan a a n w i j z i n g e n verschaffen omtrent de herkomst van de belasting. Y.Yaffe e.a., Arch.Environ Health, 38: 273-2^5, 1983

3.

Het cadmium gehalte in lever en nieren weerspiegelt de belasting van het lichaam met deze stof. Bij de mens kan met neutronen activatie analyse het cadmium gehalte in lever en nieren nauwkeurig en met weinig belasting worden bepaald.

9.

De groeiende betekenis van de Nucleaire Geneeskunde bij de bestudering van fysiologische processen maakt het noodzakelijk dat meer aandacht wordt besteed aan de toepassingsmogelijkheden van C, ^N en 1 ^0 dan nu het geval is bij de opleiding tot Nucleair Geneeskundig Specialist.

en

10. Ten onrechte wordt de invloed van verontreinigde (zure) regen op kalkrijke bodems en de daarbij behorende oeco-systemen, zoals die in Limburg algemeen voorkomen,als te verwaarlozen beschouwd.

11. Studies met behulp van biologische indicatoren doen vermoeden, dat de verontreiniging van onze atmosfeer met zware metalen voor een aanmerkelijk deel van buitenlandse herkomst is. 12. Bij de postexpositie worden gevaccineerd, belang, dat het dier wordt geobserveerd en

rabies vaccinatie kunnen minder mensen indien men meer doordrongen is van het waarmee men in aanraking is geweest, onderzocht.

13. "Totale communicatie" bevordert ook het contact tussen nietauditief gehandicapten. V4. Bij het onderwijs aan dove kinderen dienen volwassen doven te worden betrokken. 15. Jonge dove kinderen, die in een niet-gebarende omgeving opgroeien, ontwikkelen spontaan een gebaren systeem, dat structurele overeenkomst toont met de vroege taal van kinderen. S. Gol d in-Meadow, Science, 197: 401-403, 1977 Science, 221: 372-373, 1983 16. Beperking van loze woorden in spreek- en schrijftaal kan besparend werken. 17. Het idee een dam met gaten te bouwen, kan alleen ontspringen in tijden van overvloed. 18. Het predikaat "Het bier waar Limburg trots op is" dient periodiek te worden toegekend aan de brouwerij, die er het meeste recht op heeft.

Stellingen behorende bij het proefschrift: "Uptake and localization of ™mTechnetium-Methylene-Diphosphonate in bone". T.J.F. Savelkoul, Utrecht, 12 juni 1984.

UPTAKE

AND LOCALIZATION OF "mTECHNETIUM-METHYLENE-DIPHOSPHONATE IN BONE

Aan mijn ouders, Aan Marije, Rutger-Jan en Alexander

VOORWOORD Het in dit proefschrift beschreven onderzoek werd verricht in de Laboratoria van de afdeling Nucleaire Geneeskunde en de werkgroep Botstofwisseling van de Vakgroep Algemene Interne Geneeskunde. Velen hebben aan de'uitvoering van het onderzoek en het tot stand komen van dit proefschrift op direkte of indirekte wijze een bijdrage geleverd. Allen ben ik dank verschuldigd. Verder wil ik iedereen bedanken, die op enigerlei wijze aan mijn opleiding in de Nucleaire Geneeskunde, Interne Geneeskunde en Toxicologie een bijdrage hebben geleverd. Prof.dr. K.H. Ephraira was mijn opleider in de Nucleaire Geneeskunde en gaf mede de aanzet voor dit onderzoek. Dr. G. de Haas wekte mijn belangstelling voor de problematiek van de interpretatie van skeletscintigrammen in.relatie tot het botmetabolisme en heeft mij altijd weer gestimuleerd om met dit onderzoek door te gaan. Prof.dr. S.A. Duursma en dr. S.J. Oldenburg dank ik voor de stimulerende discussies en de kritische begeleiding bij dit onderzoek. Dr. W.J. Visser begeleidde de histologische experimenten, waarbij Rene Lentferink een grote inbreng had in de autoradiografische experimenten, daarbij geholpen door Peter Peters en Marja van Blokland. Rene Grouls, Jan van Ginkel, Ton Wijsman, Frank Berntsen, Dik Blok, Hans van Pelt en Frank Moerkens droegen tijdens hun bijvak radiofarmacie op eigen wijze bij aan de ontwikkeling van de gebruikte techieken en de uitvoering van de experimenten. Prof.dr. J.van de Sluys Veer en prof.dr. A. Struyvenberg wil ik danken voor mijn opleiding in de Inwendige Geneeskunde, die ik onder hun leiding mocht genieten. Prof.dr. A.N.P. van Heijst gaf mij de vrijheid om dit onderzoek af te ronden, ondanks de beperkte mankracht op het Nationaal Vergiftigingen Informatie Centrum. Dr. B. Sangster bood mij de mogelijkheid en stimuleerde mij dit proefschrift te voltooien; door zijn inzet was het mogelijk om tijdens mijn inwerkperiode binnen de afdeling Medische Toxicologie toch door te gaan met dit onderzoek. Mevr. A.E.M.I. Daalhuizen-Dur typte het manuscript en alleen dank

zij haar creativiteit en inzet was het mogelijk binnen alle tijdslimieten te blijven. Wim van Beek verzorgde de tekeningen voor dit proefschrift en Jan de Groot maakte de foto's. Het schrijven van een proefschrift, naast een drukke werkkring, vergt veel steun van een partner. Zonder jouw steun, Marije, zou het niet mogelijk zijn geweest dit te verwezenlijken. Tegen Rutger-Jan en Alexander zou ik willen zeggen: "Papa gaat weer mee voetballen".

CONTENTS 1. INTRODUCTION References 2. BONE SCINTIGRAPHY 2.1 History 2.2 Technetium -99m ( 9 ^Tc) 2.3 Gamma-Camera 2.4 Bone tissue 2.5 Uptake of " m Tc(Sn)-MDP in bone 2.6 Clinical applications 2.6.1 General principles of bone scintigraphy 2.6.2 Focal abnormalities 2.6.3 Systemic skeletal involvement 2.7 References

7 9 10 10 12 I1! 14 17 18 18 20 23 25

ELECTROLYTICALLY LABELED " m Tc-MDP: CHROMATOGRAPHY PATTERN, STABILITY AND BIODISTRIBUTION IN RATS Abstract Introduction Materials and methods Results Discussion References

33 33 34 35 37 39 44

PROTEIN-BINDING AND URINARY EXCRETION OF " m Tc-MDP AND " m Tc(Sn)-MDP Abstract Introduction Materials and methods Results Discussion References

46 46 47 47 49 54 58

THE UPTAKE OF " m Tc-PERTECHNETATE, " m Tc(Sn)-MDP, REDUCED HYDROLYZED " m T C AND 99m Tc-MDP IN FETAL RAT CALVARIA Abstract Introduction Materials and methods Results Discussion References

59 59 60 60 62 64 69

A RAPID METHOD FOR PREPARING UNDECALCIFIED SECTIONS OF B0N2 FOR AUTORADIOGRAPHIC INVESTIGATION WITH SHORT-LIVED RADIONUCLIDES Abstract Introduction Materials and methods Results Discussion References

71 71 72 72 73 7*J 77

A MICRO-AUTORADIOGRAPHIC STUDY OF THE LOCALIZATION OF 99m Tc(Sn)-MDP AND " m Tc-MDP IN UNDECALCIFIED BONE SECTIONS Abstract Introduction Materials and methods Results Discussion References

78 78 79 79 81 86 88

8 . GENERAL DISCUSSION AND SUMMARY

90

9 . SAMENVATTING

93

CURRICULUM VITAE

96

1. INTRODUCTION The clinical use of skeletal imaging agents is based on the fact that bone is a rather dynamic tissue. The search for metastatic disease remains the most important indication for bone scintigraphy. But in a lot of other diseases involving the skeleton, i t i s routinely applied. Since the labeling of polyphosphate with ™mTechnetium by Subramanian and McAfee (1) an exponential growth of bcne scintigraphy has continued to take place. A component with high skeletal affinity was combined with a radionuclide with excellent physical properties for camera imaging and a low radiation dose for the p a t i e n t . "^mTechnetium is generator produced and thus readily available in all nuclear medicine departments. Since the labeling of methylene diphosphonate with "^mTechnetium by the same investigators (2), this radiopharmaceutical has become the most widely used bone scanning agent with a rapid soft t i s s u e clearance and a high target to non-target r a t i o . Bone scintigraphy i s exceptional sensitive for detection of focal areas of increased activity, but i t is also quite non-specific. In metabolic bone diseases the entire skeleton is mostly affected with a strong influence of bone metabolism. Bone scintigraphy in these diseases however, is highly variable and the interpretation is often difficult especially with concern to the activity of the disease. For a good evaluation and quantitation of uptake of the tracer to bone, i t i s necessary to know how t h i s uptake takes place and where the tracer is located in bone. There is s t i l l a l o t of controversy about these questions. The uptake of ^'mTc-methylene diphosphonate in bone i s subject of t h i s study, with special emphasis to the localization of the tracer in bone tissue. The following items are enclosed in this study: 1. In the labeling procedure of methylene diphosphonate with 99mTechnetium always a reducing agent is involved, usually g To avoid contamination with any reducing agent and to evaluate the role of tin in the uptake, a ^'mTc-MDP complex was prepared without the use of a reducing agent. This '" m TcMDP complex is compared with "mTc(Sn)-MDP (Chapter III). 2. The protein binding and urinary excretion of ^"mTc-MDP and 99m Tc(Sn)-MDP are evaluated (Chapter IV). 3. To compare the uptake of these complexes in 'bone t i s s u e in

vitro incubation studies with fetal rat calvaria are performed (Chapter V). To study the l o c a l i z a t i o n of the complexes with autoradiography, a rapid method for preparing undecalcified bone sections with a good structural preservation had to be developed (Chapter VI). Microautoradiogr aphi c studies of the uptake of 99m Tc-MDP and "mTc(Sn)-MDP are performed (Chapter VII).

References 1. Subramanian, G., McAfee, J.G.: A new complex of Tc-99m for skeletal imaging. Radiology 99: 192-196, 1971 2. Subramanian, G., McAfee, J.G., B l a i r , R.J. et a l . : Technetium99m-methylene diphosphonate: A superior agent for skeletal imaging. Comparison with other technetium complexes. J.Nucl.Med.16: 744-755, 1975

2. BONESCINTIGRAPHY 2.1 History In 1925, a girl died after working seven years as a dial painter (1,2). Before painting the dials of watches and clocks with a radium (mesothorium) containing luminous paint, they pointed the tips of the brushes in their mouths. Anemia was the most striking symptom. At first the uptake of radioactivity was thought to be in the reticuloendothelial system (liver, spleen and bone marrow). But autoradiograms of bone sections, obtained after autopsy, showed mainly uptake in the cortical bone. This was thought to be deposited after phagocytosis by 'nistiocytes of the bone marrow. In fact, this was the first time radioactivity in human bone was recorded on a photographic plate. In 1929 Martland and Humphries concluded to deposition in bone directly, causing radiation osteitis and osteogenic sarcoma (3). In 1934 Joliot and Curie discovered the artificially induced radio-elements 1 3 N, 3°P ancj 27Si ^ ^ A l o t o f o t h e r artificially induced radionuclides are discovered since than. Chiewitz and Hevesy studied the phosphorus metabolism in rats using sodiumphosphate with 32P as tracer. Most of the tracer was located in bone tissue. Within a month however half of the radioactive phosphorus was lost. This, combined with the fluctuating ^ P excretion in urine and faeces, led to the conclusion that formation of bone is a dynarr^c process (5). Increased accumulation of ^ P at induced fracture sites in rats was first described by Bohr et al.(6). This was confirmed in humans by Tubiana in 1958 by detection of external bremsstrahlung of ^ P(7). Also a lot of studies have been performed with ^Ca and 'Ca (6,7,8). For external detection in vivo in man these radionuclides are insufficient. More possibilities supplied the strontium radionuclides. Treadwell et al. observed a high uptake of °^Sr in man in areas where new bone is being laid down (9). They concluded that this might be due (apart from vascularization, regressive local changes and so) to differences in the metabolic rate in different parts of the tumor. Photoscanning of ^ S r ^ n metastatic bone disease secondary to carcinoma of the breast, demonstrated uptake in areas of increased osteoblastic activity and Fleming et al.

10

concluded that t h i s could be used as an index of bone r e p a i r (10). °^Sr-bone imaging proved to be a r e l i a b l e and s e n s i t i v e method. However the poor resolution, low information density and high absorbed radiation dose led to the consideration of 2.8 hr). Despite differences in scanning results between °^Sr and r, due to increased blood flow in certain areas, as demonstrated by Charkes (11), ^mSr became the appropriate tracer, for use in benign diseases and in the pediatrie population. Poor target to background ratio however prevented its widespread use (7). Meanwhile some studies with radiogallium had been performed showing uptake in metastatic bone tumors and osteomyelitis. Both in osteogenic and osteolytic structures there was uptake of ? 2 Ga (12). This was morely due to uptake in tumor and infected tissues than in bone tissue. 1 Pi

Blau et a l . introduced '°F as a s k e l e t a l imaging agent and concluded t h a t the uptake in normal bone was very low in comparison to s i t e s of a c t i v e bone l y s i s or deposition (13). Van Dyke et a l . used a positron camera and evaluated the d i s t r i bution of IOF in the skeleton in r e l a t i o n to the disappearance curves in blood in r a t s and humans (14). They found i n d i c a t i o n s that the fluorine distribution in the skeleton was determined by differences in blood perfusion rate to the various bones rather than differences in extraction efficiency. The disadvantages of the strontium and fluorine radionuclides, as availability, high cost, high energy y-emitters not suitable for imaging with gamma-camera systems, has led to continuing search for other radionuclides. As alternatives the rare earths ^Sm, 171 51 135m 131 Er, Dy and a l k a l i n e e a r t h s Ba, Ba, were proved but none of them was more practical for a widespread use (7,15). In 1971 Subramanian and McAfee described the labeling of ^^mTc with tripolyphosphate (16). This radiopharmaceutical combined the ideal nuclear properties of ^^mTechnetium with the bone-seeking p r o p e r t i e s of polyphosphates. This led to a complete change of skeletal scintigraphy. Other phosphates and diphosphonates were also investigated (17-21). In 1975 the labeling of "mTc(Sn)-MDP was described (22). Labeling of ^^mTe with other phosphonates has been e s t h a b l i s h e d s i n c e then (23,24), but u n t i l l now 99mTc(Sn)-MDP i s the agent of choice for s k e l e t a l scintigraphy (25).

11

2.2 Technetium-99m "mTechnetium possesses the ideal physical properties for many of the diagnostic procedures in Nuclear Medicine. Since the l a t e sixties and early seventies i t has become common place in in vivo s t u d i e s and the use i s s t i l l growing. The ready a v a i l a b i l i t y of t h i s radionuclide i s a consequence of the development of the 99M O /99m Tc _ generator a t Brookhaven National Laboratory (U.S.A.) in 1958 (26). " m T c i s an a r t i f i c i a l radionuclide, f i r s t produced by Seaborg and Segre in 1939 (7). I t i s located in group Vll-b of the periodic table of elements, together with manganese and rhenium. In comparison with other convienently a v a i l a b l e radionuclides used in Nuclear Medicine i t has the best nuclear properties by far for imaging with the scintillation camera. The physical half l i f e i s 6.02 hours and i t has no 3-radiation. The -y-emission of 1^0 KeV (88.5$) has satisfactory tissue penetration. The 99Mo/99mTc generator consists of an alumina column to which molybdenum is absorbed. 99Mo
QQ

impurity of eluted " Tc i s "Mo. The most common other impurities consist of 1 ^ 1 I , 10^Ru and b. Fission produced 99Mo. This has the advantage of being produced c a r r i e r free. Large amounts of r a d i o a c t i v i t y can be absorbed to a relatively small alumina column. Concentration of " m T c in a small volume of saline is possible. The impurities are less and are mainly due to 99Mo, 1 3 1 I , 89 Sr and 90 Sr (28, 29). As part of the decay process of "ro^c to 9 9 Tc, extra nuclear electrons (internal conversion, Auger electrons), with energies in the range of 0.4 KeV-142.3 KeV are emitted (30). 99m Tc is eluted with normal saline from the 99Mo/99mTc generator in i t s most s t a b l e chemical s t a t e in aquous solution, 99m Tc0^ (pertechnetate), an oxoanion (valence s t a t e VII). Reduction i s

12

needed for binding to chelating agents as the bone-seeking diphosphonates. This can be achieved with a variety of reducing " M o

(67h)

. 0 45 (14%)

fij 0.87(1%)

ft,- 123 (85%)

-0 920 0 7 7 8 y, 0 739 1 372 mev

— 0 514 0

372

-0 181 0

181

;; Tc (6h)

-0 142

0 002

0 041

-0 140

"Tc ( I )

0 140 (98 4%)

0 142

(2 12 x 10'y)

Figure 1. Decay scheme of 9 9 M o (27). agents of which Sn(II)Cl2 is most commonly used. Reaction of reducing agents with other substances present can introduce unwanted radiochemical impurities. Chromatography has become the major tool for determining the radiochemical purity of a radiopharmaceutical. The most commonly radiochemical impurity is pertechnetate. In case of "'raTc-labeled phosphates and diphosphonates also reduced hydrolyzed "9m_ Technetium can be a major radiochemical impurity. The radiochemical purity of a 99m Tc radiopharmaceutical is determined by (28):

99mTc radioactivity in desired radiopharmaceutical form -x 100% Total 99mTc r a d i o a c t i v i t y present in sample A chromatographic system which is easily used in case of 99m>pc_ diphosphonates is ascending paper chromatography on Whatman 3MM 13

with acetate buffer as the eluant (3D. With this system it is possible to differentiate between reduced hydrolyzed 99m Tc (remains at the start position), pertechnetate (Rf 0.7) and the desired 9 9 m T c labeled complex (Rf 1.0).

2.3 Gamma-Camera (27,28) For the in vivo detection of " m Technetiurn in bone, the gamma camera gives the best results. Since the detection of a metastasis of a thyroid carcinoma, containing iodide, in the distal humerus by Anger in 1952 (32), the basic principles have remained essentially unchanged. The today gamma camera is often called Anger Camera. Modifications since the introduction of the Nal(Tl) crystal followed by the photomultipliers are primarily directed to increase in crystal surface area, decrease in crystal thickness, increased numbers of photomultipliers and improvements in geometrical configuration and sensitivity to light from the crystal. The solid sodium iodide crystal is activated with thallium. Pure sodium iodide crystals do not scintillate at room temperature, however, if impurities such as thallium are added, centers of luminescence are produced that can be excited at room temperature by ionizing radiation. The gamma rays enter the gamma camera through a collimator into the Nal(Tl) crystal. Depending on its energy the gamma ray will travel some distance in the crystal (several millimeters) before it transfers energy to an electron in a photo-electric or compton interaction. This will lead to light signals, absorbed by the photomultipliers. These photomultipliers consist of a photocathode and a series of 10 dynodes. Its purpose is to convert the light energy from the crystal to electrical energy and amplify the resultant pulse of electricity. The pulse height is directly proportional to the energy of the incident gamma ray. The pulses are visualized on an oscilloscope and recorded on a photographic plate. So a scintigram is performed.

2.4 Bone tissue (33, 34, 35) Bone tissue is composed of bone c e l l s and extracellular substances. It differs from other tissue not only in physicochemical

structure, but also in i t s extraordinary capacity for growth and continuous internal remodeling and regeneration. The bone structure consists of compact ( c o r t i c a l , dense) bone (80?) and spongy (trabecular, cancellous) bone (20?). Compact bone is traversed by longitudinal canals (Haversian canals), which are surrounded by a varied number of concentric lamellae. These lamellae of bone matrix, with lacunae containing bone c e l l s , and the Haversian canal constitute the Haversian system of an osteon. From the periosteal and endosteal surfaces, Volkmann's canals enter the bone at right angels and communicate with the Haversian canals. In spongy bone, the lamellae are arranged in longitudinal bundles. The trabeculae form a network, the pattern of which is influenced by the mechanical functions of i n d i v i d u a l bones. Three d i f f e r e n t c e l l types can be distinghuised, osteoblasts, osteocytes and osteoclasts. The bone e x t r a c e l l u l a r matrix consists of organic (35?) and inorganic components.

Osteoblasts Osteoblasts are located mainly at bone forming surfaces. They are responsible for synthesis and secretion of organic components, the i n t e r c e l l u l a r matrix. The o r i g i n of o s t e o b l a s t s are mesenchymal osteoprogenitor cells or pre-osteoblasts. Each osteoblast carries out a cycle of matrix synthesis after which i t either becomes burried as an internal osteocyte or becomes i n a c t i v e , but remains a r e s t i n g osteoblast or a surface osteocyte. These inactive osteoblasts and surface osteocytes assume a more flattened fibroblastic appearance and the layer of these c e l l s , connected to each other by cell processes, covers the bone surface and may act like a membrane controlling flow of compounds across the bone surface (36).

Osteocytes When osteoblasts eventually are surrounded by organic matrix (osteoid), they become osteocytes. Each osteocyte is situated in a lacuna. The osteocytes and their lacunae are larger in new bone than in older bone. An extensive canalicular system connects osteocytes with each other and with surface osteoblasts, probably serving as a channel for the flow of ions and nutrients.

15

Osteoclasts Osteoclasts are highly mobile multinucleated giant cells. They move along the bone surface, resorb bone actively and leave resorption lacunae in i t s wake. Histochemical studies show the presence of significant amounts of lysosomal and mitochondrial enzymes. On their surface exposed to bone they show a ruffled or brush border, appearing as hairlike processes extending between cell and bone. The osteoclast was long thought to be derived from the same precursor cell as the osteoblast and development from osteoblasts to osteoclasts was thought to be possible (37). An alternative view, which has acquired more evidence, is that osteoblasts and osteoclasts have distinctly different progenitor cells and that osteoclasts have their origin in migratory mononuclear phagocytes carried to sites of resorption by the blood (38).

Organic components The organic components are mainly synthesized and secreted by the osteoblasts. Collagen is secreted as procollagen and is produced by cleavage of the C-terminal and N-terminal propeptides. Bone collagen is a type I collagen and consists of two identical polypeptide chains, a^ (I) and one s l i g h t l y different chain ct2 (I)(36,39). The three polypeptide chains are held together in a helix. Multiple collagen molecules are assembled end-to-end to form fibrils, which are in turn arranged in fibers. Between the individual molecules within a fiber a gap (40 nm) e x i s t s . These gaps between the end of one molecule and the head of the next are visualized as "hole zones" on electron microscopy. Osteoblasts also synthesize and secrete other components of organic bone matrix. Osteocalcin is a protein, containing the calcium binding y-carboxyglutamic acid. The synthesis of t h i s protein is vitamin K-dependent. Osteonectin is a phospho-protein which binds to both calcium and collagen and forms a potential link between these two major constituents of bone. Other noncollagen organic constituents of bone are: sialoprotein, phosphoprotein, proteoglycans, proteolipids, lipids, peptides, structural glycoproteins and BMP (Bone Morphogenetic Protein) (40). Albumin and a2 HS-glycoprotein are devired from blood and deposited in bone at the time of mineralization (36).

16

Inorganic components Calcium and phosphorus are deposited i n i t i a l l y as amorphous s a l t s , but undergo l a t e r rearrangements into a c r y s t a l l i n e structure that resembles hydroxyapatite (Ca-jQ^O^gODH)?). Several other ions as Na+, K+, Mg2+ and CO2," can be found in the hydrate shell of bone hydroxyapatite c r y s t a l s . Mineralization takes place in the hole zones of collagen. The collagen f i b r i l itself may serve as a nucleation catalyst. Once mineralization of bone matrix i s initiated, it proceeds rapidly until 60-70% of the final amount cf mineral is deposited (primary inineralization phase). Subsequently, mineralization occurs much more slowly and may not be complete until 1-2 months later (secondary mineralization phase). The microcrystalline surface of bone mineral matrix provides a large area for exchange of ions.

2.5 Uptake of "mTc(Sn)-MDP in bone. To get a bone scintigram the tracer ""mTechnetium has to be carried to bone after i t s administration intravenously. For the transport to bone diphosphonates can be used as a c a r r i e r , because of their strong affinity to the hydroxyapatite of bone and because of their ability to pass rapidly from blood into bone (41,42). As pyrophosphate they inhibit hydroxyapatite dissolution, bone resorption and pathological calcification (41-44). The geminal phosphonates (P-C-P bound) are more efficient than the vicinal (P-C-C-P bound) phosphonates (41,45). The general formula is given in figure 2.

HO

Ri

OH

O=P—C—P=O / I \ HO

R2

OH

F i g u r e 2. General formula of geminal phosphonates. For MDP: R-, = R2 = H. Before the complexation of ^mTc with MDP, 99m Tc Q17

is reduced by

Sn(II)Cl~. After the administration of the radiopharmaceutical i t is mixed with blood and transported through the body. Equilibration with the extravascular fluid spaces and extraction of the agent by bone will take place. The main determinants affecting bone uptake are the regional blood flow, the surface area of bone mineral, the capillary permeability and the bone turnover r a t e (46,47). Some authors emphasize more the blood flow (48), while others pay more attention to the bone turnover rate (49,50), or a combined effect of extraction efficiency and blood flow (47,51). The transport of "mTc(Sn)-MDP through the capillary wall is a passive diffusion process. The uptake of the tracer in bone i s flow-limited (47,52). Only at higher flow r a t e s i t will become diffusion-limited (52,53). The rptake of the tracer in bone tissue after crossing the capillary wall is s t i l l a matter of discussion. Uptake of the labeled complex as a unit (20,54) or separated uptake of the tracer and the carrier (55-58) are both considered. For the localization of the tracer, there is increasing evidence for binding to the growing crystal surface and young hydroxya p a t i t e (20,54,56,57,59) than to uptake in the organic matrix (60). From autoradiographic studies, l o c a l i z a t i o n in bone resorbing areas (61) and cell labeling is suggested (62). Zimmer et al. (63) concluded to complexing of ^^mTc-stannous diphosphonate with alkaline phosphatase and accumulation of these complexes in areas of osteogenesis.

2.6 Clinical applications 2.6.1 General principles of bone scintigraphy. Bone scintigraphy is a sensitive method for the detection of early osseous disease especially in focal bone abnormalities. However, i t is quite non-specific and a large variety of lesior^ may lead to similar scintigraphic findings. So a l l available clinical information should be considered in the interpretation. After the administration of the radiopharmaceutical, two hours have to be waited before the scintigraphy takes place (64,65). The target to background ratio is than high enough to get images of good quality. Makler and Charkes (66) recommand a longer time delay of 4-6 hours post-injection, because a better contrast can

18

be achieved. In comparison to 99 m Tc(Sn)-pyrophosphate, 99mTc(Sn)-MDP has a higher bone uptake, significant lower blood l e v e l s and a faster urinary excretion (mean 55$, 4 hours after injection) (67). In comparison to "mTc(Sn)-HEDP, 99mTc(Sn)-MDP is also cleared more rapidly from blood, especially in the f i r s t two hours. The urinary excretion varies from 40-65^6 and is invers e l y r e l a t e d to the p a t i e n t s age. 99m-pc(Sn)_MDp bone to background ratio is significant higher than "mTc(Sn)-HEDP (68). The dose administered intravenously in adults i s in routine diagnostic studies 370 MBq (10 mCi). The absorbed radiation dose to the skeleton of adult p a t i e n t s averages 0.35 mSv/37 MBq (35 mrem/mCi). The whole body burden approximates 0.1 mSv/37 MBq (10 mrem/mCi). The dose to the kidneys i s about uwice the skeleton dose and the dose to the bone marrow i s about half the skeleton dose (28). The urinary bladder wall i s the c r i t i c a l organ. The dose to the bladder wall from the radioactive urine can be reduced s i g n i f i c a n t l y , if the bladder i s frequently emptied. Castronovo et al.(69) found a radiation dose to the bladder wall of 0.74 mSv/37 MBq for normal volunteers, 0.39 mSv/37 MBq for patients with a negative bone scan and 0.47 mSv/37 MBq for patients with a positive bone scan in the f i r s t 7 hours after administration of the radiopharmaceutical. Enhanced concentration of the tracer i s routinely found in c e r t a i n a r e a s , e.g. in t r a b e c u l a r bone more than in the diaphyseal cortex. This explains why the periarticular regions exhibit r e l a t i v e l y increased r a d i o a c t i v i t y . In c h i l d r e n , increased a c t i v i t y i s found in the growth centers, where bone turnover i s rapid. Most focal bone lesions are associated with some degree of new bone formation as well as bone destruction. The lesions appear at the scan as active ("hot"), because of the new bone formation. Fotopenic ("cold") lesions are also described (64,70-72). They can be caused by a disturbed blood supply to the particular bone area, e.g. by a compromised nutrient vessel, or by lesions with a predominant o s t e o l y t i c component and no osteogenic response. In multiple myeloma, osteol y t i c metastases, bone infarction, osteomyelitis, radiation therapy and aseptic bone necrosis, cold lesions can be found. Areas of decreased radioactivity can also be caused by protheses, pacemaker battery boxes, keys, jewels etc. In case of rapid and enhanced uptake of th'i radiopharmaceutical by pathologic bone an absent kidney sign can exist (64,73). In renal f a i l u r e no a c t i -

19

vity is seen in the kidneys, but soft tissue accumulation of the radiopharmaceutical will be impressive. Diffusely increased uptake of 99mTc(Sn)-MDP ^ n both kidneys is found in renal vascular disease and iron overload (7*0. If there i s a poor tag of Technetium increased uptake in the stomach, thyroid and salivary glands can be expected.

2.6.2 Focal abnormalities Bone tumors. Because bone scintigraphy has been shown to be highly sensitive for the early detection of skeletal metastases, it is most applied in the evaluation of patients with cancer suspected for metastases in the skeleton. The utility as a screening procedure when the patient first presents for treatment and during follow up after primary therapy, is evaluated only for a few malignant tumors. In carcinoma of the breast all patients with clinical stage III disease should have preoperative bone scans, whereas patients with stage I and II should have baseline evaluations around the time of their initial treatment (75,76). In the follow up of treatment scans can be performed every 6 months (75,77,78). A stable scan is frequently associated with clinical improvement, whereas the appearing of new other focal areas of increased uptake of radioactivity are suspect for deterioration of the disease (77,78). Citrin et al.(79,80) quantitated the uptake of radioactivity in bone metastases for assessing the response to treatment. This has not become a routine procedure. In prostate cancer preoperative scans of patients with stage I, II and III disease should be performed (75). In case of osseous metastases the scan is more sensitive than biochemical (e.g. serum acid phosphatase) parameters (81). In the follow up of treatment the scan can be of considerable value. Reduced uptake correlates closely with clinical improvement, whereas new areas of uptake reflect progressive disease. Failure to revert to normal (stable scan) does not indicate treatment failure (82-84). The bone scan may be the only method of detecting a mixed response (some metastases regress, while others advance) to therapy. Such responses are consistent with concepts regarding the polyclonal nature of prostate cancer (85).

20

In bone metastases of other malignant tumors the evaluation of bone scintigraphy is sparse. In lungtumors preoperative bone scans may be warranted even if their yield is low, because of the high operative mortality (75). In multiple myeloma "cold" and "hot" lesions may be encountered depending upon the presence of osteoblastic activity in combination with osteolytic components. Some .lesions s t a r t out scan positive and radiographic negative and later reverse this pattern as they develop (86). Of the primary malignant bone tumors osteosarcomas and the Ewing sarcomas are usually followed by bone scintigraphy (75). In osteosarcoma the bone scan appears to re; of value in determining the level of amputation (87). Intense focal uptake, more than 6 months after surgery, i s suspect for local recurrence in the stump (88). The bone scan can not differentiate between benign and malignant bone tumors. In benign bone tumors the scintigram may be useful to determine, whether the lesion is solitary or whether there are multiple foci of disease. In osteoid osteomas the scan demonstrates mostly marked activity, whereas other benign bone tumors can show variable images (89).

Infection Radionuclide bone imaging is a valuable tool for the detection of o s t e o m y e l i t i s , when c l i n i c a l complaints are poorly localized, however, falsely normal or "cold" scans may be obtained (90,91). The scan can mostly distinguish c e l l u l i t i s or soft tissue abcess from o s t e o m y e l i t i s , but differentiation of septic a r t h r i t i s is 67 often difficult (92). In conjunction with a 'Ga-citrate scan the bone scan has yet become a mainstay in the work-up of the patient with infectious disease (93). In neonatal osteomyelitis the bone scan is of l i t t l e value (9*0. The sensivity i s better with a three phase scintigraphy, especially in differentiating between septic a r t h r i t i s , soft tissue infection and osteomyelitis (95). Skeletal trauma In the evaluation of bone fractures, conventional radiography has the preference above bone scintigraphy. In patients with suspected skeletal trauma, but normal radiographs, the bone scan can detect occult fractures. For the detection of navicular and

21

f L

scaphoid fractures the bone scan is a useful means (96-98). Increased uptake of radioactivity is usually seen within 2k hours after the trauma and virtually always within three days (96). Stress fractures with normal radiographs can be detected at bone scans especially if they are multifocal (99,100). Bone seintigraphy in predicting non union or delayed union of fractures does not always differentiate between these states and normal union (101).

Paget's disease of bone (osteitis deformans) Paget's disease is characterized by focal increased bone resorption accompanied by an increased formation of new bone. The cause is unkown, but the existence of inclusion bodies in the nuclei of the abnormal osteoclasts has led to the proposal, that it may be viral in origin (102). The incidence in Europe is 3% in the population over the age of 4G years (103). The disease is in most cases clinical asymptomatic. Malignant transformation to osteosarcoma occurs in less than 1? of affected patients (102). Scintigraphy gives an accurate assessment of disease extent in active focal involvement. Those sites missed on the bone scan are metabolically inactive (104,105). The lesions seen on the bone scan and and not by X-ray correlate well with the biochemical markers (106). Response to therapy can be followed by bone scintigraphy (10^,105,107), but bone scan manifestations of recurrence are variable (108).

Aseptic necrosis Aseptic necrosis occurs if' blood supply is interrupted. In LeggPerthes disease this is characterized pathologically by infarction, necrosis and subsequent revascularization with resorption of death bone and bone repair (109). Scintigraphic findings at any site vary with the evolution of the process. Initially their is decreased uptake reversing in increased uptake, if revascularization and bone repair take place. The site of the initial scintigraphic defect is inversely correlated with the rate of healing. Any interruption of blood supply, traumatic, compressive, occlusive or metabolic can lead to bone necrosis (110-112).

22

i j \ j

;

Joint imaging The c l i n i c a l role of radionuclide j o i n t imaging has not been clearly defined. It is highly non-specific but can be helpful in assessing patients with early or atypical rheumatoid disease in whom the diagnosis i s unclear. Joint imaging with ^^mTc(Sn)diphosphonate is more sensitive than with 9"mTc-pertechnetate a s the l a t t e r localizes only in the bloodpool and/or extracellular fluid compartment. The method of diagnosing a r t h r i t i s is primairly clinical and will remain so. In degenerative joint disease the scan may be useful for detecting early stages of the disease (113). In s a c r o i l i i t i s the j o i n t uptake can be markedly increased. A sacro i l i a c uptake r a t i o should be an accurate means of diagnosing s a c r o i l i i t i s and permits early diagnosis to be made before radiologie change-' are present (11^). However, in a study of normal volunteers the uptake r a t i o s differed considerably (115).

Extra osseous localization The extra osseous accumulation of "" m Tc(Sn)-diphosphonates i s considered to be analogous to the uptake of these compounds in normal and abnormal bone. This can occur in soft tissue calcification due to a variety of diseases. The mechanism of l o c a l i zation in lesions which typically do not exhibit gross calcification is l e s s well understood. This is the fact in case of necrotic skeletal muscle or myocardium. The bone scan can help in evaluating the extent of muscle injury (116). In patients with para-osteoarthropathy the bone scan is useful to evaluate the m a t u r a t i o n of the h e t e r o t r o p i c bone. A q u a n t i t a t i v e assessment has to be made and if a s t e a d y - s t a t e plateau i s reached, surgical removal of bone can take place without recurrence (1 17,118).

2.6.3 Systemic skeletal involvement Systemic abnormality of the skeleton i s encountered in most metabolic bone diseases. Bone scanning is frequently employed in these p a t i e n t s , but in the diagnosis and evaluation of the disease activity, i t is of l i t t l e value. Yet i t is expected that

23

as the whole skeleton is involved, this must be reflected in the bone scintigram. The scintigraphic findings however are highly v a r i a b l e . Bone scintigraphy i s most extensively evaluated in patients with primary hyperparathyroidism, renal osteodystrophy and osteomalacia. For the detection and monitoring of metabolic bone diseases q u a n t i t a t i v e means are developed. In hyperparathyroidisrn, "steomalacia, renal osteodystrophy and osteoporosis, bone-to-soft tissue r a t i o (119,120,121), spine-to-kidney r a t i o (122) and whole body r e t e n t i o n of r a d i o a c t i v i t y (123,124) are evaluated. In hyperthyr oi dism t h e s e methods a r e not yet d e s c r i b e d , whereas in t h i s d i s e a s e an increased ^ate of remodeling e x i s t s with a normal amount of c a l c i f i e d bone. The scans in active hyperthyroidism can show a diffusely increased uptake. In hyperparathyroidism a so called "supersean" is rarely seen, but the bone-to-soft t i s s u e r a t i o i s increased (119,120) and the 24 hour whole body retention is also increased, 51? v.s. 19? for normals (123). Focal abnormalities are found in regions with subperiostal erosion and in brown tumors. In osteomalacia there i s also an increased bone-to-soft t i s s u e r a t i o (119,121). The 2H hour whole body retention is increased, 41 £ in the study of Fogelman et al.(123). Often there i s a f a i n t kidney v i s u a l i zation with focal areas of increased uptake. Renal osteodystrophy is a complex metabolic disorder, including osteomalacia, secondary hyperparathyroidism and osteosclerotic changes. Usually a "superscan" appearance i s present. The bone-to-soft t i s s u e r a t i o is increased (119) and the 24 hour whole body retention is markedly increased, 89? (123). In osteoporosis the scans are usually normal, except for focal increased activity at pathologic fracture s i t e s . The bone-to-soft tissue ratio is normal (119) and the whole body retention is also normal (21$ (123)). In postmenopauzal osteoporosis Fogelman et a l . (125) found significant lower values for the whole body r e t e n t i o n in women, treated with oestrogens. They found also a s i g n i f i c a n t correlation between whole body r e t e n t i o n and y ~ absorptiometry.

24

2.7 References

1. 2. 3.

Martland, H.S., Conlon, P., Knef, J.P.: Some unrecognized dangers in the use and handling of radioactive subvances. JAMA 85: 1769-1776, 1925 Martland, H.S.: Microscopic changes of certain anemias due to radioactivity. Arch.Pathol.2: 465~472, 1926 Martland, H.S., Humphries, R.E.: Osteogenic sarcoma in d i a l painters, using luminous paint. Arch.Pathol.7: 1406-417, 1929

4.

Joliot, F., Curie, I.: Artificial production of a new kind of Radio-Elements. Nature 133: 201-202, 1931*

5.

Chiewitz, 0., Hevesy, G.: Radioactive Indicators in the study of Phosphorus Metabolism in r a t s . Nature 136: 754-755, 1935 Bohr, H., Sorensen, A.H.: Study of f r a c t u r e healing by means of radio-active tracers. J.Bone Joint.Surg.32A: 567-574, 1950 Ram, P.C., Fordham, E.W.: An h i s t o r i c a l Survey of bone scanning. Sem.Nucl.Med.9: 190-196, 1979 B e l l i n , J., Laszlo, D.: Metabolism and removal of Ca -* in man. Science 117:331-33^, 1953 Treadwell, A.G., Low-Beer, B.V.A., F r i e d e l l , H.L. et al.: Metabolic studies on neoplasm of bone with the aid of radioactive Strontium. Am.J.Med.Sci.204: 521-530, 1942

6. 7. 8. 9.

10. Fleming, W.H., Mclllraith, J.D., King, E.R.: Photoscanning of Bone Lesions Utilizing Strontium 85. Radiology 77: 635-636, 1961 11. Charkes, N.P.: Some differences between bone scans made with 87m S r and 85 Sr. J.Nucl.Med.10: 491-494, 1969

12. Mulry, W.C., Dudley, H.C.: S t u d i e of r a d i o g a l l i u m as a d i a g n o s t i c agent in bone tumors. J.Lab.Clin.Med.37: 239~252, 1951 13. Blau, M., N a g l e r , W., Bender, M.A.: F l u o r i d e - 1 8 : A new Isotope for Bone Scanning. J.Nucl.Med.3: 332-334, 1962 14. van Dyke, D., Anger, H.O., Yano, Y. e t a l . : Bone blood flow shown with F ' 8 and the positron Camera. Am.J.Physiol.209: 65"70, 1965 15. O'Mara, R.E., Subramanian, G.: E x p e r i m e n t a l Agents f o r S k e l e t a l Imaging. Sem.Nucl.Med.2: 38-49, 1972 16. Subramanian, G., McAfee, J.G.: A new Complex of 99m T c f o r S k e l e t a l Imaging. Radiology 99: 192-196, 1971 17. Subramanian, G., McAfee, J.G., B e l l , E.G. e t a l . : " m T c 25

18.

19. 20.

21.

22.

23.

24.

25.

26. 27. 28. 29. 30.

31.

32. 33.

labeled polyphosphate as a skeletal imaging agent. Radiology 102: 701-704, 1972 Subramanian, G., McAfee, J.G., B l a i r , R.J. et a l . : " m T c EHDP: A potential radiopharmaceutical for skeletal imaging. J.Nucl.Med.13: 947~950, 1972 Yano, Y,., McRae, J., van Dyke, D.C. et a l . : " m T c - l a b e l e d Sn(II) Diphosphonate: A bone scanning agent. J.Nucl.Med.13: 480, 1972 Yano, Y.t McRae, J., van Dyke, D.C. et a l . : " m T c - l a b e l e d stannous ethane-1-hydroxy-1, 1-diphosphonate: A new bone scanning agent. J.Nucl.Med.14: 73-78, 1973 Castronovo, F.P., Callahan, R.J.: A new bone scanning agent. 99mTc labeled 1-hydroxy-ethylidene-1, 1-disodium phosphonate. J.Nucl.Med.13: 823-827, 1972 S u b r a m a n i a n , G., McAfee, J.G., B l a i r , R.J. e t a l . : Technetium-99tn-methylene diphosphonate: A superior agent for skeletal imaging: Comparison with other technetium complexes. J.Nucl.Med.16: 744-755, 1975 F r a n c i s , M.D., Ferguson, D.G., Tofe, A.J. et a l . : Comparative Evaluation of Three Diphosphonates: In Vitro Adsorption (1^C labeled) and In Vivo osteogenic uptake (""mTc Complexed). J.Nucl.Med.21: 1185-1189, 1980 Wang, T.S.T., Fawwaz, R.A., Johnson, G.J. et a l . : Boneseeking p r o p e r t i e s of ^" m Tc-Carbonyl Diphosphonic Acid, Dihydroxymethylene Diphosphonic Acid and Monohydroxymethylene Phosphonic Acid. J.Nucl.Med.21: 767~77O, 1980 Fogelman, I . : Diphosphonate Bone Scanning Agents - Current Concepts. Eur.J.Nucl.Med.7: 506-509, 1982 Richards, P., Tucker, W.D., Srivastava, S.C.: Technetium-99m: An h i s t o r i c a l perspective. Int.J.Appl.Radiat.Isot.33: 793~399, 1982 Early, P.J., Razzak, M.A., Sodee, D.B.: Textbook of Nuclear Medicine technology. Saint Louis, second edition, 1975 Kirchner, P.T., editor: Nuclear Medicine review syllabus. New York: Society of Nuclear Medicine, 1980 Reynolds, J.E.F., editor: Martindale The Extra Pharmacopoeia, London, 28th edition, 1982 Dillman, L.L.: Radionuclide decay schemes and nuclear parameters for use in radiation-dose estimation. J.Nucl.Med.10: 22, 1969 Owunwanne, A., Weber, D.A., 0'Hara, R.E.: Factors influencing paperchromatographic analysis of technetium-99m phosphorus compounds: Concise communication. J.Nucl.Med.19: 534-537, 1978 Anger, H.O.: Use of a Gamma-Ray Pinhole Camera for in vivo studies. Nature 170: 200-201, 1952 Leeson, C.R., Leeson, T.S.: Histology, Saunders, Philadelphia, 1966 26

34. Ham, A.W. Histology, Lippincott, Philadelphia, 1974 35. Aurbach, G.D., Marx, S.J., Spiegel, A.M.: Parathyreoidhormone, calcitonin and the calciferols. In: Williams R.H. (ed.): Textbook of Endocrinology, 6th ed. Saunders, Philadelphia, page 922-1031, 1981 36. Raisz, L.G., Kream, B.E.: Regulation of Bone Formation I. N.Engl.J.Med.3O9: 29~35, 1983 37. Rasmussen, H., Bordier, P.: The c e l l u l a r basis of metabolic bone disease. N.Engl.J.Med.289: 25~32, 1973 38. Teitelbaum, S.L., Kahn, A.J.: Mononuclear Phagocytes, Osteoclasts and bone resorption. Mineral Electrolyte Metab.3: 2-9, 1980 39. Prockop, D.J., Kiririkko, K.I., Tuderman, G. et al.: The biosynthesis of collagen and its disorders I. N.Engl.J.Med.301: 13-23, 1979 40. Urist, M.R., Delange, R.J., Finerman, G.A.M.: Bone cell differentiation and Growth factors. Science 220: 680-686, 1983 41. Fleisch, H.: Diphosphonates: History and mechanisms of action. Metab.Bone Dis.Rel.Res.4/5: 279-288, 1981 42. Fleisch, H.: Diphosphonates: Advances in Pharmacology and Therapeutics, Proceedings of the 7th International Congress of Pharmacology, Pergamon Press, Vol.3, pp.61-70, Paris, 1978 43. Fleisch, H., Russell, R.G.G., Francis, M.D.: Diphosphonates: Inhibit Hydroxyapati te Dissolution in vitro and Bone Resorption in tissue Culture and in vivo. Science 165: 1262-1264, 1969 44. Francis, M.D., Russell, R.G.G., Fleisch, H.: Diphosphonates Inhibit Formation of Calcium Phosphate Crystals in vitro and pathological Calcification in vivo. Science 165: 1264-1266, 1969 45. Jones, A.G., Francis, M.D., Davis, M.A.: Bone scanning: radionuclide Reaction Mechanism. Scan.Nucl.Med.6: 3-18, 1976

46. V a t t i m o , A., M a r t i n i , G., P i s a n i , M.: Bone u p t a k e of ""mTc-MDP in Man: I t s r e l a t i o n s h i p with local blood flow. J . N u c l . M e d . a l l . s c i . 2 6 : 173-179, 1982 47. Arnold, J . S . : K i n e t i c a n a l y s i s of bone imaging a g e n t s . I n : Colombeti L.G.: P r i n c i p l e s of Radiopharmacology, CRC, Boca. Raton, pp.205223, 1979 48. Gênant, H.K., B a n t o v i t h , G.J., Singh, M.et a l . : B o n e - s e e k i n g Radionuclides: An In vivo Study of Factors a f f e c t i n g S k e l e t a l Uptake. Radiology 113: 373"382, 1974 49. Galasko, C.S.B.: The pathological basis for skeletal scintigraphy. J.Bone Joint Surg.57"B: 353~359, 1975 50. Charkes, N.D.: Skeletal blood flow: Implications for bone27

51.

52.

53.

54. 55. 56.

57.

58.

59.

60. 61.

62.

63-

scan i n t e r p r e t a t i o n . J.Nucl.Med.21: 91-98, 1980 G a r n e t , E.S., Bowen, B.M., C o a t e s , G.: An a n a l y s i s of f a c t o r s , which influence the l o c a l accumulation of boneseeking radiopharmaceuticals. Invest.Radiol.10: 564-568, 1975 McCarthy, I.D., Hughes, S.P.F.: The r o l e of s k e l e t a l blood flow in determining the uptake of ^^ m Tc-methylene diphosphonate. Calcif.Tissue.Int.35: 508-511, 1983 S a g e r , V.V., Piccone, J.M., C h a r k e s , N.D.: S t u d i e s of S k e l e t a l t r a c e r k i n e t i c s I I I . Tc-99m(Sn)methylene-diphosphonate uptake in the canine t i b i a as a function of blood flow. J.Nucl.Med.20: 1257~1261, 1979 Bisaz, S., Jung, A., F l e i s c h , H.: Uptake by bone of pyrophosphate, diphosphonate and their technetium derivatives. Clin.Science Mol.Med.54: 265"272, 1978 Cox, P.H.: 99mTc-complexes for skeletal scintigraphy. Physicochemical factors affecting bone and bone marrow uptake. Br.J.Rad.47: 845-850, 1974 Van Langevelde H., Driessen, O.M.J., Pauwels, E.K.J. et a l . : Aspects of "^mTechnetium. Binding from Ethane-1-hydroxy-1, 1diphosphonate-""mTc-complex to bone. Eur.J.Nucl.Med.2: 47~51 , 1977 B i l l i n g h u r s t , M.W., J e t t e , D., Somers, E.: I n v e s t i g a t i o n of the i n t e r a c t i o n of h y d r o x y - a p a t i t e with technetium in association with stannous pyrophosphate. Int.J.Appl.Radiat.Isot.32: 559~566, 1981 Schumichen, C , Korfgen, T., Hoffman, G.: R e l a t i o n s h i p between complex s t a b i l i t y and biokinetics of °" m Tc-phosphate compounds. Nucl.Med.19: 7~10, 1980 F r a n c i s , M.D., Ferguson, D.L., Tofe, A.J. et a l . : Comparative evaluation of three diphosphonates: In v i t r o adsorption (C-14 labeled) and in vivo osteogenic uptake (Tc-99m labeled). J.Nucl.Med.21:11 85-1 189, 1980 Kaye, M., S i l v e r t o n , S., R o s e n t h a l l , L.: Technetium-99mpyrophosphate: studies in vivo and in v i t r o . J.Nucl.Med.16: 40-45, 1975 C h r i s t e n s e n , S.B., K r o g s g a a r d , O.W.: L o c a l i z a t i o n of "' m Tc-methylene diphosphonate in epiphysial growth p l a t e s of rats. J.Nucl.Med.22: 237-245, 1981 G u i l l e m a r t , A., Besnard, J - C , Le Pape, A. e t a l . : S k e l e t a l uptake of pyropphosphate l a b e l e d with Technetium-95m and Technetium-96, as evaluated by autoradiography. J.Nucl.Med.10: 895~899, 1978 Zimmer, A.M., I s i t m a n , A.T., Holmes, R.A.: E n z y m a t i c inhibition of diphosphonate: A proposed mechanism of t i s s u e uptake. J.Nucl.Med.16: 352-356, 1975

28

' _ ' ' '

^

64. Muroff, L.R.: Optimizing the performance and interpretation of bone scans. Clin.Nucl.Med.6: 68-76, 1981 65. Potsaid, M.S., Guiberteau, M.J., McKusick, K.A.: Quality of bone scans compared with time between dose and scan. J.Nucl.Med.18: 787"789, 1977 66. Makler, P.T., Charkos, N.D.: Studies of s k e l e t a l t r a c e r k i n e t i c s IV. Optimum time delay for Tc-99m(Sn) methylene diphosphonate bone imaging. J.Nucl.Med.21: 641-645, 1980 67. Rudd, T.G., Allen, D.R., H a r t n e t t , D.E.: Tc-99m-methylene diphosphonate versus Tc-99m pyrophosphate: Biologic and Clinical comparison. J.Nucl.Med.18: 872-876, 1977 68. Rudd, T.G., Allen, D.R., Smith, F.D.: Technetium-99m-labelëd methylene diphosphonate and hydroxyethylidine diphosphate: Biologic and Clinical comparison. J.Nucl.Med.20: 821-826, 1979 69. Castronovo, F.P., McKusick, K.A., Strauss, H.W.: Bladder wall dosimetry after the administration of ^9m T c _ d i p h o S pj l o n a t e > Health physics.40: 744-746, 1981 70. Goris, M.L., Lawrence, V.B., Etcubanas, E.: Photopenic lesions in bone scintigraphy. Clin.Nucl.Med.5:299-301, 1980 71. Spencer, R.P., S z i k l a s , J.J., Rosenberg R. et a l . : Hemivertebral "disappearance" on bone scan. J.Nucl.Med.22: 454-456, 1981 72. Sy, W.M., Westring, D.W., Weinberger, G.: "Cold" lesions on bone imaging. J.Nucl.Med.16: 1013-1016, 1975 73. Sy, W.M., Patel, D., Faunce, H.: Significance of absent or faint kidney sign on bone scan. J.Nucl.Med.16: 454-456, 1975

74. Koizumi, K., Tonami, N., H i s a d a , K.: D i f f u s e l y " m Tc-99m-MDP uptake in both kidneys. Nucl.Med.6: 362-365, 1981

increased

75. McNeil, B.J.: Rationale for the use of bone scans in selected metastatic and primary bone tumors. Scan.Nucl.Med.8: 336-345, 1978 76. Hammond, N., Jones, S.E., Salmon, S.E. et al.: Predictive value of bone scans in an adjuvant breast cancer program. Cancer 41: 138-142, 1978 77. Bitram, J.D., Bekerman, C , Desser, R.K.: Predictive value of serial bone scans in assessing response to chemotherapy in advanced breast cancer. Cancer 45: 1562-1568, 1980 78. Citrin, D.L., Hougen, C , Zweibel, W.et al.: Use of serial bone scans in assessing response of bone metastases to systemic treatment. Cancer 47: 680-685, 1980 79. Citrin, D.L., Bessent, R.G., Tuohy, J.B. et al.: Quantitative bone scanning: A method for assessing response of bone 29

80.

81.

82.

83.

metastases to treatment. Lancet 1: 1132-1133, 1971» C i t r i n , D.L., Eessent, R.G., McGinley, E. et a l . : Dynamic studies with ^^mTc-HEDP in normal subjects and in patients with bone tumors. J.Nucl.Med.16: 886-890, 1975 Shafer, R.B., Reinke, D.B.: Contribution of bone scan, serum acid and alkaline phosphatase, and radiographic bone survey to management of newly diagnosed carcinoma of the prostate. Clin.Nucl.Med.2: 200-203, 1977 Langhammer, H., Sintermann, R., Hor, G. et a l . : S e r i a l bone scintigraphy for assessing effectiveness of treatment of osseous metastases from prostatie cancer. Nucl.Med.17: 87~91, 1978 F i t z p a t r i c k , J.M., Constable, A.R., Sherwood, T. et a l . : Serial bone scanning: Assessment of treatment response in carcinoma of the prostate. Br.J.Urol.50: 5 5 5 - 5 6 1 , 1978

84. P o l l e n , J . J . , S h l a e r , W.J.: O s t e o b l a s t i c response to successfull treatment of metastatic cancer of the prostate. A m.J.Roentgenol.132:927-931, 1979 85. Pollen, J.J., Gerber, K., Ashburn, W.G. et a l . : Nuclear bone imaging in metastatic cancer of the prostate. Cancer 47: 2585-2595, 1981

86. Woolfenden, J.M., P i t t , M.J., D u r i e , G.M. e t a l . : Comparison of bone scintigraphy and radiography in m u l t i p l e myeloma. Radiology 134: 723"728, 1980 87. Hughes, S.P.F., Benson, M.K.D., E l l , P.J. e t a l . : Use of 9b>mTe-EHDP a s s c a n n i n g agent in d e t e c t i o n of metastases from osteosarcoma. Fortsch.Geb.Roentgenstr.Nuklearmed.126: 551-555, 1977 88. McKillop, J.H., E t c u b a n a s , E., G o r i s , M.L.: I n d i c a t i o n s f o r and l i m i t a t i o n s of bone s c i n t i g r a p h y in osteogenic sarcoma: Review of 55 p a t i e n t s . Cancer 48: 1133-1138, 1981

89. G i l d a y , D.L., Ash, J.M.: Benign bone tumors. Sem.Nucl.Med.6: 33~46, 1976 90. Wald, E.R., M i r r o , R., G a r t n e r , J . C . : P i t f a l l s i n t h e diagnosis of acute o s t e o m y e l i t i s by bone scan. Clin.Pediatr.19: 597-600, 1980 91. B e r k o w i t z , I.D., Wenzel, W.: "Normal" t e c h n e t i u m bone scans in p a t i e n t s with acute o s t e o m y e l i t i s . Am.J.Diss.Child.134: 828-830, 1980 92. H o w i e , D.W., S a v a g e , J . P . , W i l s o n , T.G. e t a l . : The t e c h n e t i u m phosphate bone scan in t h e d i a g n o s i s of o s t e o m y e l i t i s in childhood. J.Bone J o i n t Surg.65-A:431-437, 1983 93. Handmaker, H., Leonards, R.: The bone scan in i n f l a m m a t o r y osseous d i s e a s e . Sem.Nucl.Med.6: 95-105, 1976 91. Ash, J.M., G i l d a y , D.L.: F u t i l i t y of bone s c a n n i n g i n neonatal o s t e o m y e l i t i s : Concise communication. 30

J.Nucl.Med.21: 417-420, 1980 95. Maurer, A.H., Chen, D.C.P., Camargo, E.E. et a l . : U t i l i t y of three-phase skeletal seintigraphy in suspected osteomyelitis. J.Nucl.Med.22: 941-949, 1981 96. Matin, P.: Appearance of bone scans following f r a c t u r e s including immediate and long-term studies. J.Nucl.Med.20: 1227~1231, 1979 97. Ganel, A., Engel, J., Oster, Z. et a l . : Bone scanning in assessment of fractures of the scaphoid. J.Hand.Surg.4: 540-543, 1979 98. Marty, R., Denney, J.D., McKamey, M.R. et a l . : Bone trauma and related benign disease: Assesment by bone scanning. Sem.Nucl.Med.6: 107-120, 1976 99. Roub, L.W., Gumerman, L.W., Hanley, E.N- et a l . : Bone s t r e s s : Radionuclide imaging perspective. Radiology 132: 431-438, 1979 100.Meurman, K.O.A., E l f r i n g , S.: S t r e s s f r a c t u r e in s o l d i e r s : Multifocal bone disorder; comparative radiologie and s c i n t i graphic study. Radiology 134: 483"387, 1980 101.Silberstein, E.B.: Nuclear o r t h o p e d i c s . J.Nucl.Med.21: 997"999, 1980 102.Kanis, J.A., Evanson, J.M., R u s s e l l , R.G.G.: Paget's d i s e a s e of bone. Diagnosis and management. Metab.Bone Dis.Rel.Res.4/5: 219-230, 1981 103.Barker, D.J.P.: The epidemiology of Paget's disease. Metab.Bone Dis.Rel.Res.4/5: 213-234, 1981 1 04.Seraf i n i , A.N.: Paget's d i s e a s e of bone. Sera.Nucl.Med.6: 47'58, 1976 105.Fogelman, I., Carr, D., Boyle, I.T.: The r o l e of bone scanning in Paget's disease. Metab.Bone Dis.Rel.Res.4/5: 243-254, 1981 1 06.Khairi, M.R.A., Wellman, H.N., Robb, J.A. et a l . : Paget's disease of bone ( o s t e i t i s deformans): Symptomatic lesions and bone scan. Ann.Intern.Med.79: 348-351, 1973 107.Goldman, A.B., Braunstein, P., Wilkinson, D. et a l . : Radionuclide uptake s t u d i e s of bone: A q u a n t i t a t i v e method of evaluating the response of patients with Paget's disease to diphosphonate therapy. Radiology 117: 365~369, 1975 108.Vellenga, C.J.L.R., Pauwels, E.K.J., Bijvoet, D.L.M, et a l . : S c i n t i g r a p h i c aspects of the recurrence of t r e a t e d Paget's disease of bone. J.Nucl.Med.22: 510-517, 1981 109.Danigelis, J.A.: Pinhole imaging in Legg-Perthes d i s e a s e : Further observations. Sera.Nucl.Med.6: 69-82, 1976 110.Lotke, P.A., Ecker, M.L., Abass, A.: Painful knees in older patients: Radionuclide diagnosis of possible o s t e o n e c r o s i s with spontaneous resolution. J.Bone Joint Surg.59A: 617~621, 1977 31

11 I.Gregg, P.J., Walder, D.N.: Seintigraphy versus radiography in early diagnosis of experimental bone necrosis: with special reference to caisson disease of bone. J.Bone Joint Surg.62B: 214-221, 1980 112.Burt, R.W., Matthews, T.J.: Aseptic Necrosis of the Knee: Bone scintigraphy. Am.J.Roentgenol.138: 571-573, 1982 113.Hoffer, P.B., Gênant, H.K.: Radionuclide Joint Imaging. Sera.Nucl.Med.6: 121-137, 1976 114.Domeij-Nyberg, B., Kjallman, M., Nylen, 0. et al.: The r e l i a b i l i t y of quantitative bone scanning in s a c r o i l i i t i s . Scand.J.Rheumatol.9: 77~79, 1980 115.Vyas, K., Eklem, M., Seto, H. et a l . : Q u a n t i t a t i v e s c i n t i graphy of s a c r o i l i a c j o i n t s : Effects of age, gender and laterality. Am.J.Roentgenol.136: 589~592, 1981 116.Hoflin, F., E l l i , P.J., Rosier, H. et a l . : Acute muscular pain and positive bone scan. Nucl.Med.Communications 3: 30-34, 1982 117.de Haas, G., Hoekstra, A., Bakker, H.: P a r a - o s t e o a r t r o p a t h i e bij patiënten met traumatische afwijkingen van het centrale zenuwstelsel; onderzoek en waardering van de ectopische botvorming met behulp van het radiopharmacon "" m Tc-pyrofosfaat. Ned.T.Geneesk.121: 1289-1292, 1977 1i8.Tanaka, T., Rossier, A.B., Hussey, R.W. et a l . : Q u a n t i t a t i v e assessment of p a r a - o s t e o - a r t h r o p a t h y and i t s naturation on s e r i a l radionuclide bone image. Radiology 123: 217-221, 1977 1 1 9.Rosenthall, L., Kaye, M.: Technetium-99m-pyrophosphate kinetics and imaging in metabolic bone disease. J.Nucl.Medf.16: 33~39, 1975 120.Rosenthall, L., Kaye, M.: Observations on the mechanism of 99 m Tc-labeled phosphate complex uptake in metabolic bone disease. Sem.Nucl.Med.6: 59-67, 1976 121 .Fogelman, I., McKillop, J.H., Bessent, R.G. et a l . : The r o l e of bone scanning in osteomalacia. J.Nucl.Med.19: 245-248, 1978 122.Holmes, R.A.: Quantification of S k e l e t a l Tc-99m labeled phosphates to detect metabolic bone disease. J.Nucl.Med.19: 330-331, 1978 123.Fogelman, I., Bessent, R.G., Turner, J.G. et a l . : The use of whole body retention of Tc~99m diphosphonate in the diagnosis of metabolic bone disease. J.Nucl.Med.19: 270-275, 1978 124.Martin, W., Fogelman, I., Bessent, R.G.: Measurement of 24hour whole body retention of Tc~99m HEDP by a gamma camera. J.Nucl.Med.22: 542-545, 1981 125.Fogelman, I., Bessent, R.G., Cohen, H.N. et a l . : S k e l e t a l uptake of diphosphonate. Method for p r e d i c t i o n of postmenopausal osteoporosis. Lancet I I : 667"67O, 1980 32

3. ELECTROLYTICALLY LABELED 99mTcMDP:CHROMATOGRAPHIC PATTERN, STABILITY AND BIODISTRIBUTION IN RATS.

T.J.F. Savelkoul, S.J. Oldenburg, W.J. van Oort, S.A.Duursma.

Abstract The l a b e l i n g of methylene diphosphonate with ^^mTechnetium i s possible after reduction of pertechnetate by means of controlled potential electrolysis. This results in a "^mTc-MDP complex which differs slightly from ^^mTc(Sn)-MDP in paper and gel chromatography. The s c i n t i g r a p h i c images of both preparations are comparable in quality. B i o d i s t r i b u t i o n in r a t s shows a higher bone uptake for the 99mTc(Sn)-MDP complex, whereas ^^mTc-MDP shows a higher uptake in the gastric wall.

The I n t e r n a t i o n a l Journal of Applied Radiation and Isotopes, 1984, in press.

33

Introduction Since the l a b e l i n g of diphosphonates with 99m T e c h n e t i u m (1-3), skeletal scintigraphy i s a routine procedure in the evaluation of bone l e s i o n s . The most widely used agent i s 9 9 m p e c n n e t i u m _ Methylene-diphosphonate (99mxc-MDP) (J|). For the labeling procedure i t is necessary to reduce pertechnetate to a lower oxidation s t a t e , Tc(IV), in the presence of excess MDP. As a reducing agent, stannous chloride i s commonly used. However, the role of tin in the in vivo distribution, the mechanism of uptake and the l o c a l i z a t i o n of techneti um-methylenediphosphonate i s not clear. For this reason other reducing agents were tried. Using concent r a t e d HC1 and vacuum evaporation, i t was possible to prepare technetium complexes with the a n t i - o x i d a n t s ascorbic acid and gentisic acid (5). Autoclaving 9"mTc0^ with HC1 for 30 minutes at 121°C resulted in a reduction, as controlled with paper chromatography. A technetium-hydroxy-ethylidene-diphosphonate (99nijc_ HEDP) complex could be formed after reduction of 99mTeO- with concentrated HBr adding HEDP after evaporation (7). Reduction with NaBHjj and subsequent complexing with HEDP, led to a mixture of different "mTc(NaBHi4)-HEDP complexes (8). To avoid contamination of the radiopharmaceutical with any reducing agent electrolytical reduction of 99mTcO- in presence of the desired ligand might be possible. Russel (9) labeled tetracycline and EDTA with "mTechnetium in this way. In an electrochemical cell, consisting of two compartments connected by a s a l t bridge, Steigman et al.(10) prepared several "^mTechnetium radiopharmaceuticals with reduction of ""mTc0^ by hydrogen evolved at the cathode. As "mTc(Sn)-MDP i s the most widely used complex, we studied the possibility of preparing ™mTc-MDP after electrolytical reduction of " m Tc0jj in presence of excess MDP. The notation "mTc(Sn)-MDP refers to the "^mTc-complex prepared by Sn(II) as the reductant and "mTc-MDP refers to the electrochemically prepared complex. To evaluate the r o l e of t i n with respect to the bone uptake and s t a b i l i t y of the complex, t h i s ^^mTc-MDP complex has been compared with ^^mTc(Sn)-MDP. S t a b i l i t y and composition of the complexes prepared were investigated by paper and gel chromatography. The in vivo d i s t r i b u t i o n of both preparations has been studied in rats.

Materials and Methods Equipment Controlled potential electrolysis was carried out with a homemade potentiostat. A 5 ml polarographic cell (Metrohm EA 88O-T-5) was employed with a mercury pool e l e c t r o d e (area 7.1 cmr) an Ag/AgCl-reference electrode (Ingold 3f3~9O), and a platinum wire a u x i l l i a r y e l e c t r o d e . The p o t e n t i a l of the working electrode was -1.25 V vs Ag/AgCl-reference electrode. Ascending paper chromatography was performed on Whatman 3 MM with 0.5 M a c e t a t e buffer (pH 5.0) as the eluant. The r a d i o a c t i v i t y was detected by means of a thin-layer scanner (Berthold Scanner I I Lb 2722-2) with a s o l i d s c i n t i l l a t i o n (Nal) d e t e c t o r . Gel chromatography was performed on Biogel P4 (Biorad) 100-200 mesh with normal s a l i n e as the eluant in 0.9 x 28 cm and 0.9 x 1 3 cm columns. Radioactivity was counted in a gamma counter with a well-type Nal c r y s t a l (Tri Gamma Baird Atomic). A c t i v i t i e s injected into the rats were measured in a dose calibrator. Scintigrams of rats were made with a Pho IV gamma camera (Searle). Tissue radioactivities were counted in the gamma counter. Chemicals Methylenediphosphonate was obtained commercially (Sigma Chemical Company). 99mTcQ- was eluted with normal saline from a generator and diluted as needed. All other reagents were of normal analyt i c a l reagent grade. Animals Male Wistar r a t s weighing 180-230 gram were used. Procedures " Tc-MDP was prepared as follows. MDP (acid form) was dissolved in normal s a l i n e . The pH was adjusted to 7.0 by adding NaOH. " m Tc0^ was added to this solution. The MDP concentration in the working solut.on was 0.H%. Of this solution 1.5 ml was put in the e l e c t r o l y s i s vessel. The s o l u t i o n was deaerated by a stream of nitrogen for about 15 minutes. This was continued during the electrochemical experiment. Samples were taken at t = 0', t=30', t=60' and t=90'. The samples were analyzed by paper and gel chromatography.

35

99mTc(Sn)-MDP w a s prepared by d i s s o l v i n g 200 mg MDP and 50 mg SnClp^Hp 0 in 90 ml normal s a l i n e . The pH was adjusted to 7.0 by NaOH and normal saline was added to a total volume of 100 ml. One m i l l i l i t e r of t h i s solution was mixed with 2 ml 99mTcO-_ Of both p r e p a r a t i o n s chromatography on paper and Biogel was performed. Gel chromatography of the peak fraction, considered as the 99mTc_MDp c o m p i e x w a s repeated on another column of the same gel phase.

*r o

is

on

1

05

J?

as

;»

j

j ,

; ;

iii

^-y-

j S

J i

10

>i

Figure 1. Radioactivity peaks of '^mTc~MDP on scanned paper chromatograms at t = 0', t = 60' and t = 90' from top to bottom respectively.

36

Both preparations were diluted with normal saline (1:100 and 1:500) and samples were analyzed by paper chromatography. A scintigram was performed on four rats two hours after injection of 0.5 ml of the radiopharmaceuticals (500 pCi; 18.5 MBq) in the tail vein. Tissue distributions, at 1.5 hour and 3 hours after injection were performed In 23 rats injected with 1.59 ± 0.03 MBq of either radiopharmaceutical.

Results During the electrolysis at certain time intervals, 5 yl aliquots were analyzed by paper chromatography. The results at t=0', t=60' and t = 90' are given in figure 1. At t = 0' all radioactivity was detected at Rf = 0.6-0.7, the Rf value of " m T c 0 ^ . At t = 60' about k0% of the radioactivity was detected at Rf 0.6-0.7, and 60? at Rf 0.9. At t = 90' only 20? was found at Rf 0.6-0.7 and 80? at Rf 0.9. In table 1 the Rf values of the different " m T c compounds are given with the peak radioactivity fractions obtained from gel chromatography. " m T c 0 2 (Rf=O) was not detected.

Table 1. Rf values of the different 99m Tc-compounds by paper and g e l chromatography. The peak f r a c t i o n s eluated from the gel columns (13 x 0.9 cm and 28 x 0.9 cm) a r e a l s o g i v e n ; h y d r o l y z e d 99m Tc i s not e l u a t e d from t h e s e columns. Components

hydrolyzed " m T c 99m

Rf value

Peak F r a c t i o n 13 x 0.9 cm 2 8 x 0.9 cm

0 0.6 - 0.7

18

34

Tc(Sn)-MDP

1.0

6

10

Tc-MDP

0.9

7

13

99m 99rn

Tc0^

37

In figures 2 and 3 the gel chromatographic patterns of 99mTc_MDp are given at t=15' and t=90'. They are identical on both columns. The peak fractions are again analyzed on paper and gel chromatography (figures 4a and 4b). Nearly a l l a c t i v i t y was detected in the same fractions (no.13 and no.17).

Table 2. Biological distribution of "raTc-MDP and "mTe(Sn)-MDP in r a t s . "mTc-MDP (peak fraction 13, f i g . 3) (% dose/g tissue)

" m Tc(Sn)-MDP dose/g tissue)

1 .5 hr after dose n=7

3 hr a f t e r dose n=6

1.5 hr a f t e r dose n=5

3 hr a f t e r dose

femur

1 • 99±O .12

1.63+0 31

3 .49±0 .71

3.24+0. 50

femur muscle

0 .03+0 .00

0.02+0 01

0.02+0 . 0 2

0.02+0. 02

blood

1 .16+0 . 0 2

0.09+0 01

0 04+0 .01

0.02+0. 00

liver

0 .25±0 .05

0.21±0 07

0. 51±0 .08

0.39+0. 10

spleen

0 .11+0 .04

0.06+0 03

0. 45+0 . 1 0

0.52+0. 20

kidney

0 .35+0 .03

O.33±O. 04

0. 50±0 .14

O.39±O. 04

gastric wall

0 .76+0 .25

0.34+0. 09

0. 02+0 .01

0.02+0. 00

Dilutions of both preparations gave different r e s u l t s . In a dilution of 1:100, ^^mTc(Sn)-MDP showed a significant amount of a hydrolyzed form of technetium. When diluted 1:500 nearly a l l 99mTc(Sn)-MDP disappeared and the amount of hydrolyzed ^^mTc increased. In contrast ^"mTc-MDP remains s t a b l e even in a dilution of 1:500. The scintigraphic images of both preparations are comparable in quality (figures 5a and 5b). A clear uptake of r a d i o a c t i v i t y in the skeleton was obtained with l i t t l e uptake in the soft tissues. The b i o d i s t r i b u t ion in r a t s for both preparations are given in table 2. For both preparations a clear bone uptake is observed. The uptake of "mTc(Sn)-MDP is higher than that of "mTc-MDP. The uptake of 99mTc(Sn)-MDP j s higher in liver and spleen.

38

a

2

*

&

B

ID 12

1* 16

19 10 11 2
Figure 2. Gel chromatography of "mTc-MDP after 15 minutes (top) and 90 minutes e l e c t r o l y s i s (bottom); colutnn^J x 0.9 cm, peak 99m TcO, fraction 7 = "mTc-MDP and fraction 18 Discussion E l e c t r o l y s i s of y99m. ' TcO^-solutions in a mercury pool gave only s u f f i c i e n t y i e l d at e l e c t r o d e p o t e n t i a l s of -1.2 V vs Ag/AgClr e f e r e n c e e l e c t r o d e s , or more n e g a t i v e . As already found by Russel (9,11), the p o t e n t i a l in t h i s t r a c e r experiment i s 0.3 0.5 V more negative than found in most polarographic experiments at carrier concentration range. Russel found a half wave potent i a l of 0.7 vs SCE at pH 7.8, in a s o l u t i o n of 0.1 M EDTA. Claessens (12) found -0.84 V vs SCE a t pH 7.3 in a s o l u t i o n of 99 and MDP (1:4). These and other data on polarographic

39

.'

O

U «

\

Vt 1b 18 2U 22 2-i 26 28 10 32 J i 56 38 40

Figure 3. Gelchromatography of ^^mTc-MDP after 15 minutes (top) and 90 minutes e l e c t r o l y s i s (bottom); column 28 x 0.9 cm, peak fraction 13 = " m T c - M D P and fractions 32 "TcO,

p r o p e r t i e s of " % c 0 ^ were obtained by use of mercury drop e l e c t r o d e s , w i t h drop t i m e s of 1 - 5 s e c , w i t h c a r r i e r conc e n t r a t i o n s of TcOT i n t h e same o r d e r of magnitude as t h e complexing agents. Electrochemical reduction of compounds with a mercury pool e l e c t r o d e d i f f e r s from t h a t w i t h a mercury drop e l e c t r o d e in surface area a c c e s s i b i l i t y , time window, refreshment of the accessible electrode surface and adsorption of parent compounds and reaction products. Furthermore, during the prolonged coulometric experiments, traces of oxygen have more time

O

«f

1

H

O

o

3

10 11 1-. :o

'3

2C J2 2-

?o 2S

0

2

<.

(.

8

10 12 1H 16

1S 20

22 14 2o 2&

JS J « J ) JÜ Jv 1 J

Figure 4a/b. Gel and paper chromatograms of peak fraction 7 as described in figure 2 (4a) and of peak f r a c t i o n 13 as described in fig.3 (4b); column 13 x 0.9 cm, there i s only one obvious peak, considered as "^mTc-MDP.

to i n t e r f e r e with the e l e c t r o d e - p r o c e s s and may influence the r e v e r s i b i l i t y of the redox reactions. This may occasionally lead to high activation energies, expressed as half-wave potentials, as shown by the TeOh reduction. The evaluation of the e l e c t r o l y s i s products i s p o s s i b l e with paper chromatography. Using sodium acetate (pH 5) as the solvent, QQm

"~

QQm

a good separation between ""'TcO^ and ""Tc-MDP can be achieved with hydrolyzed technetium remaining at the start position (13). The Rf value of " m Tc-MDP differs slightly from the Rf value of

Figure 5a/b. Scintigram of a rat 2 hourt. i. ter injection of ""m-MDP, peak fraction 13 as described in fig.3 (5a) and after injection of "mTc(Sn)-MDP (5b). " m Tc(Sn)-MDP (0.9 vs 1.0). After 90 minutes of electrolysis about 80^ of the radioactivity is concentrated at Rf 0.9. No hydrolyzed technetium is detected by then. With gel chromatography and ^^ m Tc-MDP are very well separat-ed (1H-16). There is only one obvious ^^mTc-MDP peak with both columns used. Repeated gel chromatography of the peak r e vealed no obvious pertechnetate or hydrolyzed technetium (as established by paper chromatography). Again the peaks of "mTc(Sn)-MDP differ slightly from the "mTc-MDP peaks (fraction 6 vs fraction 7 and fraction 10 vs fraction 13 on the respective

columns). The 99mTc-MDP c o m p i e x Was in v i t r o more s t a b l e to d i l u t i o n than the 99mTc(Sn)-MDP complex. Schumichen (17,18) established a fast transformation in a hydrolyzed technetium compound after dilution of " m Tc-pyrophosphate and "mTc-HEDP with neutral s a l i n e , especially with concerning 9 ^ m Tc-pyrophosphate. For °9mTc-complexes prepared without reducing agents a higher s t a b i l i t y against a i r oxidation has been suggested (10). The in vivo uptake as evaluated on the scintigrams is comparable in quality for both preparations. However, the biodistributions show more bone uptake for the 9"mTc(Sn)-MDP complex, whereas 99mTc_MDp ghc^g m o r e uptake in the g a s t r i c wall. This may be caused by a reoxidation to "mTc0]j in vivo. Russel (9,11) proved by means of reversed pulse polarography under certain conditions that a reoxidation of reduced technetium i s possible in v i t r o . The observations of Russel in his in vitro experiment and our in vivo results are in agreement so far. In conclusion: I t is possible to prepare a ^9mjc_MDp c o m p i e x by electrolytical reduction of technetium pertechnetate in presence of excess MDP. The chromatographic properties of this complex on paper and gel chromatography are s l i g h t l y different from y m " Tc(Sn)-MDP. The bone-seeking properties of both complexes are comparable. Tin i s not necessary for bone uptake. The s t a b i l i t y of 9"mTc-MDP to d i l u t i o n in v i t r o i s higher but in vivo a r e oxidation to pertechnetate may occur to some extent.

References 1.

2.

3.

4.

5.

6. 7.

8.

9.

10.

11. 12. 13.

14. 15.

Subramanian, G., McAfee, J.G., B l a i r , R.J. et a l . : 99mTC-EHDP: A p o t e n t i a l radiopharmaceutical for s k e l e t a l imaging. J.Nucl.Med.13: 947"95O, 1972 Yano, Y., McRae, J., Van Dyke, D.C. et a l . : Technetium-99mlabeled stannous Ethane-1-hydroxy-1-diphosphonate: a new bone scanning agent. J.Nucl.Med.14: 73"78, 1973 Subramanian, G., McAfee, J.G., B l a i r , R.J. e t a l . : Technetium-99m-methylene diphosphortate. A superior agent for skeletal imaging: comparison with other technetium complexes. J.Nucl.Med.16: 744-755, 1975 Fogelman, I.: Diphosphonate bone scanning agents - Current concepts. Eur.J.Nucl.Med.7: 506-509, 1982 Sundrehagen, E.: Formation of reduced 99m-j>c ^y concentrated hydrochloric acid and vacuum evaporation. Int.J.Appl.Radiat.Isot.33: 89~91 , 1982 De Liverant, J. and Wolf, W.: Studies on the reduction of (99m Tc ) Tc0 - b y hydrochloric acid. Int.J.Appl.Radiat.Isot.33: 857-860, 1982 Deutsch, E., Libson, K., Becker, C.B. et a l . : Preparation and b i o l o g i c a l d i s t r i b u t i o n of technetium diphosphonate radiotracers synthesized without stannous ion. J.Nucl.Med.21: 859-866, 1980 Pinkerton, T.C., Ferguson, D.L., Deutsch, E. et a l . : In vivo distributions ofsome component f r a c t i o n s of "^TcCNaBH^)EHDP mixtures separated by anion exchange high performance liquid chromatography. Int.J.Appl.Radiat.Isot.33: 907"915, 1982 Russel, CD.: C a r r i e r e l e c t r o c h e m i s t r y of p e r t e c h n e t a t e : a p p l i c a t i o n to radiopharmaceutical labelling by controlled potential electrolysis at chemically inert electrodes. Int.J.Appl.Radiat.Isot.28: 241-249, 1977 Steigman, J., Chin, E.V. and Solomon, N.A.: S c i n t i p h o t o s in rabbits made with Tc-99m preparations reduced by electrolysis and by SnCl^: Concise communication. J.Nucl.Med.20: 766-660, 1979 Russel, CD.: Electrochemistry of Technetium. Int.J.Appl.Radiat.Isot.33: 883-339, 1982 Claessens, R.A.M.: Technetium and tin diphosphonates. Thesis Nijmegen, 1982 Owunwanne, A., Weber, D.A. and 0'Hara, R.E.: Factors influencing paper chromatographic analysis of Technetium-99m phosphorus compounds: Concise Communication. J.Nucl.Med.19: 534-537, 1978 Billinghurst, M.W. and Palser R.F.: Gel chromatography as an analytical tool for 99m.j.c radiopharmaceuticals. J.Nucl.Med.15: 722-723, 1974 Johannson, B.: Behaviour of 99mi- c _ labe ^ eC j
sephadex and Biogel. J.Nucl.Med.16: 1987~1088, 1975 16. Van de Brand, J.A.G.M.: Technetium ( t i n ) Ethane - 1 - Hydroxy1, 1-Diphosphonate complexes. Thesis Utrecht, 1981 17. Schumichen, C, Mackenbrock, B. and Hoffman, G.: Kinetics of various ^^mTc-Sn-Pyrophosphate compounds in the r a t . I I . In v i t r o studies. Nucl.Med.16: 157-162, 1977 18. Schumichen, C , Hohloch, M. and Hoffman, G.: Complexing of reduced Technetium and Tin ( I I ) by c h e l a t i n g Phosphate compounds, II In v i t r o s t a b i l i t y of Pyrophosphate and Ethane1, Hydroxy-1, Diphosphonate (EHDP) complexes. Nucl.Mea.18: 105-109, 1979

4. PROTEIN-BINDING AND URINARY EXCRETION OF 99mTc-MDP AND 99mTc(Sn)-MDP.

T.J.F. Savelkoul, J.J. van Ginkel, .R.J.E. Grouls, S.J. Oldenburg, S.A. Duursma.

Abstract In an incubation experiment the Human Serum Albumine (HSA) binding of "mTc-MDP (electrolytically labeled) and "mTe(Sn)-MDP i s e s t a b l i s h e d . During the incubation some pertechnetate i s formed and in case of ^^mTc(Sn)-MDP also some hydrolyzed 99m Technetium. The HSA binding of "mTc-MDP is less than the HSA binding of "mTc(Sn)-MDP as established with gel chromatography, TCA-precipi t a t i o n , ammonium sulfate precipitation and u l t r a f i l tration. TCA-precipitation seems to be an insufficient method for determining the protein binding of "mTc(Sn)-MDP. The urinary excretion in rats shows only one "°mTc-compound in both cases. The bone seeking properties of the urine excreted ""mTc-compound are confirmed in another rat.

Submitted for publication.

Introduction In s k e l e t a l scintigraphy Wm7c(Sn)-MDP i s the agent of choice in comparison with other bone-seeking radiopharmaceuticals (1). The s t a b i l i t y of t h i s complex depends on the MDP concentration (24). In vivo the radiopharmaceutical is diluted in plasma. Protein binding e x i s t s and may prevent h y d r o l y s i s or o x i d a t i o n (3-6). The e x t e n t t o which p r o t e i n - b i n d i n g occurs i s h i g h l y v a r i a b l e . Using p r e c i p i t a t i o n with t r i c h l o r o a c e t i c acid, the protein bound f r a c t i o n of " m Tc(Sn)-MDP in blood v a r i e d from 14$ (7) t o 30? (1,8). With p r e c i p i t a t i o n by ammoniumsulfate, Saha et a l . found 125S p r o t e i n - b i n d i n g (7), whereas Schumichen e t a l . found 22.4? (9). D i a l y s i s in s a l i n e r e s u l t e d in a p r o t e i n bound f r a c t i o n of 1 &% in t h e experiment of Saha e t a l . (7). From gel f i l t r a t i o n measurements Johanssen e t a l . concluded t o a p r o t e i n bound f r a c t i o n of H5% (10). In e l e c t r o p h o r e t i c s t u d i e s of p r o t e i n binding in plasma ^^mTc(Sn)-MDP was mostly bound and transported by human serum albumin (8,11). In a r e c e n t study we showed t h a t the e l e c t r o l y t i c a l l y l a b e l e d 99m T c _ M D p w a s m o r e s t a b l e t 0 d i l u t i o n than " m Tc(Sn)~MDP. However, in vivo oxidation to ""mTc0]j occurred t o some extent (12). The aim of t h i s study i s t o e s t a b l i s h the p r o t e i n binding of " m Tc(Sn)-MDP and " m Tc-MDP in v i t r o , using four d i f f e r e n t methods, and t o e v a l u a t e the u r i n a r y e x c r e t i o n of both r a d i o pharmaceuticals in r a t s .

Materials and Methods Equipment Ascending paper chromatography was performed on Whatman 3 MM with 0.5 M a c e t a t e buffer (pH 5.0) as the e l u a n t . Gel chromatography was performed on Biogel P4 (Biorad) 100-200 mesh with rormal saline as the eluant in 0.9 x 28 cm and 0.9 x 60 cm columns (Pharmacia). For U I t r a f i l t r a t i o n a S t i r r e d c e l l of 65 ml was used with a D i a f l o U l t r a f i l t e r PM10 (cut off >10,000 mol.wt) (Amicon B.V., Oosterhout, Holland). Radioactivity was counted in a gamma counter with a well-type Nal crystal (Tri Gamma Baird Atomic).

Scintigrams of rats were made with a Pho IV gamma camera (Searle). Chemicals. Methylene diphosphonate (acid form) was obtained commercially (Sigma Chemical Company). 99mTcO- w a s e i u t e d with normal saline from a generator (Byk-Mallinckrodt) and diluted as needed. Human Serum Albumine (HSA 20$) was obtained from the Central Laboratory of the Bloodtransfusion Service (Amsterdam). All other reagents were of normal analytical reagent grade. Animals Male Wistar r a t s of 250 gram and male and female Wistar r a t s of 180 gram were used. Procedures "9mTc-MDP anci 99mTc(Sn)-MDP were prepared as described previously (12). The MDP concentration of the working solution in the elect r o l y s i s was 1.5%. During the e l e c t r o l y s i s samples were controlled with paper chromatography and after 90 minutes also with gel chromatography (column 0.9 x 28 cm). A p r e p a r a t i v e s e p a r a t i o n was performed on a column of 0.9 x 60 cm of the same gel phase. The MDP concentration of both radiopharmaceuticals was adj usted to 0.3?. A sample of 25 yl of both radiopharmaceuticals was incubated with 30 ml of a HSA s o l u t i o n of 3-5? at 37°C. During the incubation 5 ul samples were taken at t=0', t = 1 0 \ t = 2 0 \ t = 3 0 \ t = 4 0 \ t=50' and t=60'. The samples were analyzed with paper chromatography. The percentage albumin binding after one hour of incubation was determined using four different methods: 1. Gel chromatography; a sample of 0.2 ml was analyzed on a 0.9 x 28 cm column. 2. Precipitation with trichloroacetic acid (TCA); a sample of 100 yl was added to 1.0 ml TCA 5% and mixed forcedly; the precipitated albumine was separated by centrifugation (10 minutes, 3000 rpm) and an aliquot of 25 pi of the supernatant was counted for radioactivity; this was compared with a standard prepared by adding 100 pi of the incubated solution to 1.0 ml normal saline, handled in the same way. 3. P r e c i p i t a t i o n with ammonium s u l f a t e ; a sample of 100 yl was added to 1.0 ml ammonium s u l f a t e 55i£ and processed in the same

way as the TCA precipitation. U. Ultrafiltration;

the rest of the incubated solution '+ 25 ml)

was put into the ultraf iltration cell;

the solution was

brought

under nitrogen pressure (2.1 0^ kPa) and stirred continuously; ultrafiltrate was collected

the

in 2 ml fractions, of which s-i.T.ples

of 200 ^1 were taken for counting the radioactivity, and compared

to a standard of 200 ul of the incubated solution.

I ri the

plateauphase of the ultraf iltration a paper chromatogran

z-C t.ne

ultrafiltrate was performed. The urethra of four male

Wistar

rats f?50 gr a.ti! was ligatec

subcutaneously. Two hours after injection cf 0.^ ml .' 15.5 M5C m

" Tc-MDP

(2 rats) and of

99m

of

Tc(Sn)-MI)? (2 rats) in the tail

vein, a scintigram was performed. The rats were sacrified and the content of the bladder aspirated in a syringe. Samples (-, _1: cf the aspirate were analyzed on paper and gel chromatcgraphy. ~r.e rest was injected

in the tail vein of another rat •' 1=" gram'.

Two hours later scintigrams of these rats were perfor.xeu.

Results

P a p e r and g e l c h r o m a t o g r a m s of ^^ m Tc-MDP a f t e r 9C m n u * e s of e l e c t r o l y s i s a r e g i v e n i n f i g u r e 1. In t h e gel chrs.-natogram only o n e peak of r a d i o a c t i v i t y i s found a t f r a c t i o n 1 3 . H o w e v e r , on t h e paper chromatogram one can d i s t i n g u i s h two peaks 'Rf 0.8 and 1.0). I n t h e p r e p a r a t i v e s e p a r a t i o n on c o l u m n 0.9 x 6? cm :i 1 s o one peak of r a d i o a c t i v i t y i s found ( f r a c t i o n 2 4/25). A n a l y s i s cf s a m p l e s from t h i s f r a c t i o n w i t h paper chromatography a l s o g i v e s two peaks a t t h e same p o s i t i o n s as in f i g u r e 1. The paper ar.a gel chromatograms of "" m Tc(Sn)-MDP showed t h e p r e s e n c e of o n l y one peak. I n f i g u r e 2 t h e paper chromatograms a t t = 0', t = 1 0 \ t = ? 0 ' , t = ?0', t=iJO', t=50' and t=60' of both i n c u b a t i o n e x p e r i m e n t s a r e shown. I n t h e i n c u b a t i o n of "" m Tc-MDP t w o p e a k s can be d i s t i n g u i s h e d w i t h Rf 0 . 7 - 0 . 9 . HSA s h o w s t h e s e same Rf v a l u e s a s was o b s e r v e d from t h e n i n h y d r i n e r e a c t i o n and U-V a b s o r p t i o n . The sequence of t h e p a p e r c h r o m a t o g r a m s s h o w s a s h i f t of t h e r a d i o a c t i v i t y t o w a r d s Rf 0.8-0.9. Paper chromatography a f t e r ' incubat ion of HSA w i t h 99 m Tc(Sn)-MDP O n l y shows r a d i o a c t i v i t y in t h e f i r s t chromat o g r a m ( t = 0') a t Rf 0 . 7 - 0 . 9 . H o w e v e r , in t h e s e q u e n c e of t h e s e c h r o m a t o g r a m s i n c r e a s i n g r a d i o a c t i v i t y i s d e t e c t e d a t Rf 0

and a t Rf 0.7.

(reduced hydrolyzed

0.5

Rf O

i

0

I

i

i

i

|

i

l

l

l

l

i

0J& 0.7

i

l

i

i

|

i

I

t

i

|

i

l

l

2 U 6 8 10 12 U 16 18 20 22 2i 26 28 30 32 3t 36 35 iO ^2 W i 6 i.8 50 Figure 1. Paper and gel chromatogram of " m Tc-MDP after 90 minutes of electrolysis.

50

In figure 3 the gel chromatograms of albumine binding of 99mTc_MDP a n d 99mTc(Sn)_MDp a r e g i v e n > i n both some pertechnetate exists. The binding percentages for both incubations as established with the different methods are given in table 1. The binding is calculated as percentage of total radioactivity. The albumine binding is greater for ^^mTc(Sn)-MDP with all methods used. For gel chromatography, ammonium sulfate-precipitation and u l t r a f i l tration, the difference i s in the same order of magnitude. In the TCA-precipitation however a large difference in albumine binding is determined.

Table 1. HSA-binding of "mTc-MDP and

99m

Tc(Sn)-MDP

Methods

99mTc_MDP

" m Tc(Sn)-MDP

Gelchromatography

41.0 + 3.9

47.8 ± 1.6

T.C.A.-precipitation

50.6 + 3.0

80.3 ± 6.8

Ammoniumsulfate-precipitation

43.7 + 5 . 0

56.8 ± 5.8

Ultrafiltration

54.4 + 4 . 3

59.9 ± 3.3

The plateau phases of the u l t r a f i l t r a t i o n are shown in figure 4. Paper chromatography of the u l t r a f i l t r a t e in the plateau phase showü radioactivity at Rf 0.9 and Rf 0.6-0.7 for "mTc-MDP and at Rf 1.0 and Rf 0.6-0.7 for "mTc(Sn)MDP (figure 5). The scintigrams of the rats two hours after injection of either radiopharmaceutical show a high uptake in the bladder and normal uptake in the skeleton. The paper chromatograms of the urine show only one radioactivity peak, for "mTc-MDP and "mTc(Sn)-MDP, at Rf 0.8 and Rf 1.0 r e s p e c t i v e l y . Gel chromatography of the urine shows also one peak at the position, where 9™mTc-MDP and "mTc(Sn)-MDP are to be expected respectively. Injection of the urine in another rat again results in a bone scintigram for both radiopharmaceuticals (figure 6).

51

B5 0 6 0 7 CB CS 10

Rf

0

t =60

Fi gure 2. Paper chromatograms during the incubation of 9"mTe-MDP (left) and " m Tc(Sn)-MDP (right) with HSA at 37°C.

52

i r 0 2 4

. . ii—i—i—i—i—i—i—i—i—i—i—i—r"r-1—r—i—i—i 8 10 12 14 16 8 20 2? 1U 26 26 X V 34 .16 38 iC 42 U. 4G iB 50

Fi &ure 3» Gel 'chromatogram of " m Tc-MDP (top) and " m Tc(Sn)-MDP (bottom) a f t e r one hour of i n c u b a t i o n w i t h HSA.at 3 7 ° C The peak a t f r a c t i o n 8 i s c o n s i d e r e d as t h e H S A - " m T c - c o m p l e x peak. Some pertechnetate (fraction 4) i s detected.

53

8'J -

GO -

20 10 -

0

8

'O

'2

U

"ê

20

22

24

26

28

30

Fi gure H. Plateau phase of u l t r a f i l t r a t i o n of ^^mTc-MDP • • • and 99mTc(Sn)-MDP Percentage binding on the abcis. Fraction numbers on the ordinate.

Discussion The electrolytical reduction of ^'"TcOjj is performed in a MDP concentration of 1.5?. This results in a ^^mTc-MDP complex with a different behaviour on paper chromatography in comparison to our e a r l i e r experiments where a MDP concentration of QA% was used (12). The two peaks of r a d i o a c t i v i t y t h a t now can be d i s t i n guished, suggest a mixture of at least two ''mTc-MDP complexes. Essential is that on gel chromatography only one peak of radioa c t i v i t y i s e s t a b l i s h e d with no p e r t e c h n e t a t e . On the paper chromatogram of the urine only one peak of r a d i o a c t i v i t y was detected at Rf 0.8. I n j e c t i o n in another r a t r e s u l t s in a bone scintigram, so i t can be concluded, that t h i s ^^mTc-com pound with clear bone seeking properties is a ™mTc-MDP com piex. ™mTc(Sn)MDP is excreted in urine in the same way with maintenance of the bone seeking p r o p e r t i e s . Van den Brand (13) found 3 hours a f t e r injection of "mTc(Sn)-HEDP 98% of the radioactivity in urine of r a t s excreted as "mTc(Sn)-HEDP.

Rf O

0.5 _J

0.6 I

0.7 I

O.B l

0.9 l

1.0

Rf O

C.5

0.6

0.7

0.6

0.9

1.0

Figure 5. Paper chromatograms of the ultrafiltrate of and " m Tc(Sn)-MDP (bottom).

55

99m

Tc-MDP (top)

i •r

Figure 6. Scintigram of a rat two hours after injection of the urine of another r a t injected two hours before with ""mTc-MDP. During the incubation of ""mTc-MDP with HSA the radioactivity is detected in the region, where HSA i s expected on paper chromatography. In the sequence of chromatograms a shift i s seen towards Rf 0.9. No a c t i v i t y i s d e t e c t e d at Rf 0. The gel chromatogram at the end of the incubation period shows a p a t t e r n with an estimated protein binding of 41? together with some pertechnet a t e . I t i s nnt l i k e l y t h a t t h e r e i s mere p e r t e c h n e t a t e present in the beginning than at the end of the incubation. This i s most probably caused by conversion to the faster migrating ""mTc-MDP complex. The paper chromatograms of ^^mTc(Sn)-MDP during the incubation with HSA show reduced hydrolyzed "°mTechnetium, which i s formed together with the appearance of radioactivity at the Rf of pertechnetate. In the experiments of Owunwanne et al. (5) and Saha et al.(6) no augmentation of hydrolyzed 9 9 m T e G i i r i e t i um a n d QQm

__

yy

TcOjj was noticed. The MDP concentration however, was considerably higher than in our experiments, whereas i t is in good 56

agreement with the expected in vivo concentration range. The HSA binding of "mTc-MDP is slightly less than "mTc(Sn)-MDP. Gel chromatography, ammonium s u l f a t e p r e c i p i t a t i o n and u l t r a f i l t r a t i o n are comparable in the calculated HSA binding. The TCAp r e c i p i t a t i o n r e s u l t s in . considerably higher HSA binding for "mTc(Sn)-MDP. This might be caused by decomposition of the 99mTc(Sn)-MDP complex under strongly acid conditions and subsequent binding of hydrolyzed 99mjc t 0 t ! i e (jenaturated proteins. Comparison of TCA- and ammonium sulfate precipitation in proteinbinding of "mTc(Sn)-MDP and "mTc(Sn)-HEDP resulted in a higher binding value in ease of TCA-precipitation (7,9). In comparison with other studies the "mTe(Sn)-MDP HSA binding is higher in our study considering the p r e c i p i t a t i o n r e a c t i o n s . The binding value determined by gel chromatography is comparable to Johannsen et al.ClO). In conclusion: The HSA binding of "mTc-MDP i s l e s s than "mTc(Sn)-MDP. For "mTc-MDP the binding varied from 111-51»*, whereas the binding for 99mTc(Sn)_MDp v a r i e d frOm 111-60? except for the TCA-precipitation (80*). The methods of gel chromatography ammonium s u l f a t e p r e c i p i t a t i o n and ultrafiltration are comparable in the results whereas TCA-precipitation is not a good method for evaluating the protein-binding of phonate complexes. Both radiopharmaceuticals are excreted in the urine with maintenance of their bone seeking properties.

57

i

:

References 1.

2. 3.

4. 5.

6.

7. 8.

9.

10.

11. 12.

13.

Subramanian, G., McAfee, J.G., B l a i r , R.J. e t a l . : Technetium-99m-methylene diphosphonate: A superior agent for skeletal imaging: Comparison with other technetium complexes. J.Nucl.Med.16: 744-755, 1975 Claessens, R.A.M.: Technetium and tin diphosphonates. Thesis Nijmegen 1982 Schumichen, C , Korfgen, T., Hoffmann G.: R e l a t i o n s h i p between Complex S t a b i l i t y and Biokinetics of ^^mTc-phosphate Compounds. Nucl-Med.19: 7~10, 1980 Khan, R.A.A.: Factors in fluencing the d i s t r i b u t i o n of99mtechnetium methylene diphosphonate in bone and soft tissues. Experientia 3^: 1598-1600, 1978 Owunwanne, A., O'Mara, R.E., O'Brien, C : The s t a b i l i t y of ' m Tc phosphorus compounds in plasma both in vivo and in vitro. J.Radioanal.Chem.59: 571-578, 1980 Sana, G.B., Boyd, CM.: Heart S t a b i l i t y of " m T c - R a d i o pharmaceuticals on 37°C. Int.J.Appl.Radiat.Isotop.7: 337~339, 1980 Sana, G.B., Boyd, CM.: A Study of p r o t e i n - b i n d i n g of " m T c Methylene Diphosphonate in Plasma. Int.J.AppLRadiat.Isotop.6: 201-206, 1979 Bunting, R.W., Mieto, 0., Callahan, R.J. et a l : Plasma and serum binding of technetium-99m Methylene Diphosphonate. J.Nucl.Med.23: P76, 1982 Schumichen, C , Koch, K., Kraus, A. et a l . : Binding of Technetium-99m to plasma proteins: Influence on the D i s t r i bution of Tc~99m Phosphate Agents. J.Nucl.Med.21: 1080-1085, 1980 Johannsen, B., Berger, R., Schomacker, K.: The binding of Technetium compounds by human serum Albumin. Radiochem.Radioanal. Letters 42: 177-188, 1980 Vanlic-Razumenic, N., Petrovic, J., Gorki*, J.: Binding of Tc~99m-MDP complex by human blood serum constituents. J.Lab.Comp.Radiopharm.19: 1568-1569, 1982 Savelkoul, T.J.F., Oldenburg, S.J., van Oort, W.J. et a l . : Electrolytically labeled °"mTc-MDP: Chromatographic Pattern, Stability and Biodistribution in Rats. Int.J.Appl.Radiat.Isotop. 1984 (in press) Van de Brand, J.A.G.M.: Technetium (tin) Ethana-1-Hydroxy-1, 1-Diphosphonate complexes. Thesis Utrecht, 1981

58

5. THE UPTAKE OF 99mTc-PERTECHNETATE, 99mTc(Sn)-MDP. REDUCED HYDROLYZED 9 9 and 99mTc-MDP IN FETAL RAT CALVARIA.

T.J.F. Savelkoul, R.J.E. Grouls, S.J. Oldenburg, S.A. Duursma.

Abstract

Fetal r a t calvaria were clamped in an incubation chamber with a buffered s a l t solution. Administration of "mTc(Sn)~MDP did not result in any uptake of radio-activity in te calvaria. The uptake of reduced hydrolyzed ^^mTc was about 20? after two hours. For 99mTc-MDP ^ n e Uptake was about H0% after two hours, while no h y d r o l y s i s was observed. The uptake of "'mTc-MDP as a unit i s very l i k e l y , whereas h y d r o l y s i s of ""mTc(Sn)-MDP i s necessary before bone uptake.

Submitted for publication.

59

Introduction In bone abnormalities with active bone formation, bone fractures, Paget's disease of bone, osteomyelitis, metastatic or primary bone tumors and some metabolic bone diseases, the uptake of 99mTechnetium labeled diphosphonates or polyphosphates is higher in comparison with normal bone (1-6). This is not only caused by a higher local blood flow, but also by an increase in extraction efficiency (7). The mechanism of uptake and the s i t e of binding in bone i s s t i l l a matter of controversy. Uptake of the labeled complex as a unit (8,9) or a separate uptake of the tracer with only a carrier function for the ligand is considered (10-13). For the binding in bone, there i s more evidence for a r e l a t i o n to hydroxyapatite (8,9,11,12), especially the growing crystal surface (14), than to collagen (15). The aim of this study is to evaluate the uptake of different QQm 7 Tc-compounds in bone. In an incubation experiment with f e t a l r a t c a l v a r i a t h e u p t a k e was s t u d i e d of " m T c 0 ] j , 9 9 m Tc(Sn)-MDP, reduced hydrolyzed " m T c and " m Tc-MDP ( e l e e t r o l y t i c a l l y labeled) d u r i n g t w o h o u r s a f t e r a d m i n i s t r a t i o n of t h e s e r a d i o pharmaceuti c a l s .

Materials and Methods Preparation of "" m Tc-compounds. ^" m Tc0^ i s e l u t e d w i t h normal s a l i n e from a g e n e r a t o r (Byk M a l l i n c k r o d t ) . 18.5 MBq " m T c 0 ^ i s mixed w i t h Hank's Balanced Salt Solution, pH 7.H (Gi bco Europe) to a volume of 100.0 ml. SnMDP: 25.0 mg SnCl 2 .2H 2 0 (Merck) d i s s o l v e d in H N HC1, 100.0 mg MDP (Sigma Chemical Company) and Hank's Balanced S a l t S o l u t i o n (H.B.S.S.) added t o a volume of + 95 m l , pH i s a d j u s t e d t o 7.4 by NaOH and H.B.S.S. added t o a t o t a l volume of 100.0 ml. " m Tc(Sn)-MDP: 18.5 MBq " m T c 0 ^ i s mixed with Sn-MDP t o a t o t a l volume of 10.0 ml. Reduced h y d r o l y z e d " m T c : 10 ul Sn-MDP i s mixed w i t h 18.5 MBq 99 m Tc0^. After 15 m i n u t e s s t a n d i n g , H.B.S.S. i s added t o a t o t a l volume of 10.0 ml. " m Tc-MDP i s prepared in a three e l e c t r o d e system with a mercury pool electrode, an Ag/AgCl-reference e l e c t r o d e (Ingold 373~9O) and a platinum wire a u x i l l i a r y e l e c t r o d e , as described previous60

ly (16). The MDP concentration in the electrolysis vessel is 1.0%. After the electrolysis 9 9 m T c _ M D p i s separated from the remaining 9Qm ^ with g e l chromatography on Biogel Pk ( B i o r a d ) , 100-200 mesh, w i t h normal s a l i n e as t h e e l u a n t in a 0.9 x 28 cm g l a s s column. 18.5 MBq " m Tc-MDP (350 . 1 ) i s d i l u t e d with 5.68 x 10~ 3 M MDP in H.B.S.S. (1 mg MDP/ml), pH l.H t o a t o t a l volume of 10.0 ml. Ascending paper chromatography of a l l '" m Tc-compounds i s p e r formed on Whatman 3 MM with 0.5 M a c e t a t e buffer (pH 5.0) as t h e eluant.

Incubation experiment Modified Ussing chambers are used for the incubation experiment (17). Fetal calvaria of Wistar r a t s on the 19th day of gestation are wedged in a rubber ring between the two parts of the incubat i o n chamber (figure 1). The volume of compartment A i s 600 .-1, the volume of compartment B is ^00 - 1 . The calvaria are directed with their convex s i t e to compartment B. After placing the calvaria, compartment A is f i l l e d with 600 pi Hank's Balanced Salt Solution (pH 7.^, temperature 37°C). The incubation chamber i s placed in a waterbath of 37°C. Before f i l l i n g compartment B, 30 minutes a r e waited to control if t h e r e i s any leakage from A to B. When no leakage is observed, ^00 ^1 of either ^^mTc-compound is put into compartment B. During the incubation the solution in compartment B is mixed by an air flow of 2.5 ml min""1. At t = 0 \ t = 5 \ t = 10', t = 20', t = 3 0 \ t = 4 5 \ t = 6 0 \ t=75', t = 9 0 \ t = 105' and t = 1 20', 5 pi samples are taken from B. These samples are counted for radioactivity in a gamma counter with a well-type Nal c r y s t a l (Tri Gamma Baird Atomic). From s i x samples a paper chromatography i s immediately performed (t = 0', t = 10', t = 30', t = 60', t = 90', t = 120'). These chromatograms are cut in pieces of 1 cm that are counted in the same gamma counter. At t=125'• a sample from the original solution is analyzed in the same way. Ten ul samples from compartment A are taken at t = 15', t = 35', t = 65', t = 1 10' and t = 125', counted for r a d i o a c t i v i t y and analyzed with paper chromatography in the same way as the samples from compartment B. In case of 99mTc_MDp t 0 0 u t t l e radioactivity was counted in compartment B for analysis with paper chromatography. 61

All data are corrected for radioactive decay. For the samples of compartment A, a l l values are corrected for volume difference with compartment B.

Results The percentages of 99m Tc0 JJ in incubation media and the quality control are presented in table 1. The uptake of 9 9 m T c O - is nil, if corrected for diffusion to compartment A. The paper chromatograms from samples of both comparments show only one peak of radioactivity at Rf 0.7. The chromatogram of the original solution after 125 minutes gives the same pattern. All radioactivity is detected at the Rf of " m T c 0 ^ . The uptake of " m Tc(Sn)-MDP gives the same result as " m T c 0 ^ (table 2). If corrected for the radioactivity appearing in A, there is no uptake in the calvaria. The complex, detected at Rf 1.0 is stable

Figure 1. Incubation chamber. A=compartment A (600 u l ) ; B=compartment B (400 y l ) . 1= f e t a l r a t calvarium;2= a i r flow system; 3= rubber r i n g ; 4= perspex. Both p a r t s a r e screwed t i g h t l y together. 62

during the incubation in compartment B. However, paper chromatography of samples from compartment A show also radioactivity at Rf 0.0 indicating t h a t , to some extent, hydrolyzed 99m>pc j S formed- The chromatogram of the o r i g i n a l solution after 125 minutes shows only radioactivity at Rf 1.0.

10

20

X time (mm I

Figure 2. Bone uptake of the ""mTc-compounds in calvaria. Left axis: percentage of administered r a d i o a c t i v i t y in the. incubation medium. + + + = " m T c 0 ^ ; ooo = "mTc(Sn)-MDP;DDn = reduced hydrolyzed " m T c ; x-x-x = "mTc-MDP. The uptake of reduced hydrolyzed ^"mTc in the calvaria is about 20? after two hours of incubation (table 3). The ^^mTc appearing in compartment A is reoxidized to "" m Tc0^ to some extent. The chromatogram of the original solution after 125 minutes shows a pattern with 95? of the radioactivity at Rf 0.0 and 5% at Rf 0.7. The uptake of ^^ m Tc-MDP in the calvaria is about iJO? after two hours (table JJ). This complex is stable during the incubation period and has a Rf value of 0.9 (16). Hardly any diffusion of radioactivity into compartment A can be detected. The paper chromatogram and gel chromatography of the original solution after 125 minutes, shows only one peak of radioactivity at the place of 9 9 m T c _ M D P <

63

Figure 2 shows the uptake of the

99m

Tc-compounds relative to each

other.

Discussion To study the influx and efflux of ions i n t o , respectively from bone t i s s u e , incubation of fetal cal varia in a buffered s a l t solution is an accepted method (17-19). In comparison with the in

Table 1. Percentages of 99mTcO- i n i ncu bation media and quality control on Whatman 3 MM. (5 cal varia)

Compartment (x ± s . d . ) Time

B

A+B

A

Rf-value 0

0.7

1.0

-

100

-

(minutes) 1

100.0

100.0

5

100.1+0.8

100.2+0.8

10

100.1+0.6

100.2+0.6

-

100

-

99.8+0.9 99.5+0.6

-

100

-

-

100

-

-

100

-

-

100

-

-

1' 0

-

-

100

-

-

100

-

-

100

-

0.1

15 20

99.6+1.1

30

98.8+1.4 0.9+1.5

35 45

98.1±1.8

99.4+0.8

60

97.9+1.2

99-3+0.8 1.6+1.5

65 75 90

96.0+2.6

no 120 125

99.6+2.0 3.5+1.6

95 105

99.8+1 .4

97.5+1.7 96.6+2.6

100.0+1.9 4.5+1.6 100.4+1.9

95.2+2.2 5.6+1.7

64

vivo uptake of 9 9 m T c _ d i p h o S p . n o n a t e s t n e i n i t a l phase of d i s appearing of r a d i o a c t i v i t y from blood i s omitted here. The memb r a n e s e n v e l o p i n g t h e c a l v a r i a r e m a i n i n t a c t in our method. In f e t a l c a l v a r i a the m i n e r a l i z a t i o n p r o c e s s i s not completed yet and bone formation i s s t i l l continuing. At places of bone formation an excellent uptake i s seen of 99m Tc a f t e r a d m i n i s t r a t i o n of 99m Tc-diphosphonates (14). No uptake of " m T c 0 ~ could be observed in our s t u d y as was e x p e c t e d , but some passage t h r o u g h t h e

T a b l e 2. P e r c e n t a g e s of 99mTc(Sn)-MDP in i n c u b a t i o n media and q u a l i t y control on Whatman 3 MM. (TO c a l v a r i a ) Compartment (x +

Time

B

A

Rf-value

s.d.)

A+ B

0

0.7

-

-

1

• -J

(minutes) 0

100.0

100.0

5

99.5±1.1

99.6+1 .2

10

99.2+1.6

9 9 . 5 + 1 .6

20

98.9+1.7

9 9 . 5 + 1 .6

30

98.1+1.6

99.4+1 •5 1 . 4+0 .4

35 45

97.3±1-3

99.4+1 .0

60

96.8+1.3

9 9 . 8 + 0 .8

3. 4 + 0 .9

65 75

96.0+1.5

100.0+1 • 3

90

9 4 . 5 + 1 .6

9 9 . 3 + 1.5

5. 0+1 .0

95

125

1 00

i

1 -

-

100

43

-

57

1 -

-

loo :

28

-

72

-

-

100

21

-

79

-

-

100

6. 1+0 .8 99.5+1 . 2

92.9+1.5

6. 9 + 0.9

65

1

'

9 9 . 3 + 1 .3

9 3 . 5 + 1 .5

110

120

-

0 . 5 + 0 .3

15

105

100

14

8

cal varia from compartment B to A arose. The same r e s u l t s are found after incubation with "mTc(Sn)-MDP, although there i s passage through the calvaria to A in the same order as for As is stated before (10,11,13), i t i s possible that MDP has only a carrier function for 99mT. t o j ^ e anc j the uptake into bone is independent for MDP. Schumichen (13) found a dissocation of 99mTc(Sn)-MDP after dilution, with a subsequent formation of a reduced hydrolyzed ^^mTc-compound. After injection in r a t s this

Table 3. Percentages of reduced hydrolyzed 9 " m Tc in incubation media and quality control on Whatman 3 MM. (10 calvaria) Compartment (x .t s . d . )

Time

B

A+B

A

Rf-value

0

0.7

1.0

(minutes) 0

100.0

100.0

5

95.3+1.9

95.U+1.9

10

92.8+2.1

93.0+2.1

20

90.7+2.8

91.0+2.8

30

88.6+3.2

88.9+2.9 0.5+0.2

35 45

86.6+3.1

87.5+3.3

60

84.5+3.1

85.7+3.1 1.4±0.3

65 75

82.4+3. i:

84.2+3.2

90

81.0+3-4

83.1+3.3 2.3+0.4

95

125

3

97

-

3

100

-

-

100

-

-

98

2

-

31

69

-

95

5

-

26

74

-

93

7

-

18

82

-

81.5+3.2

78.7+3.3 3.010.6

110 120

-

0.2±0.1

15

105

97

77.4+3.2

80.9+2.9 3.6+0.5

66

hydrolyzed 99mTc h a d n o bone-seeking properties and was concentrated in the kidneys. In a biodistribution study in r a t s , Khan (20) also concluded to a higher uptake in the kidneys, after a 1000-fold dilution of ^^mTc(Sn)-KDP. However an even higher uptake was seen in the thyroid, which was ascribed to a r e oxidation of reduced 99mTc(IV) t o pertechnetate. In our study reduced hydrolyzed ^mlc was produced by a 1000-fold dilution in Hank's Balanced Salt Solution.

Table 4. Percentages of ^^mTc-MDP in media and quality control on Whatman 3 MM. (6 calvaria)

Compartment (x :t s.d.)

Time

B

A

Rf-value

0

0.7

0.9

-

-

100

93.5 + 3• 3 88.6 + 1 .8

-

-

100

80.1+1 .8 77.5+3 .0

-

-

100

71 .7+2 .5 69.0+2 .5

-

-

100

68.1+2 .8 66.5±2 .5

-

-

100

-

-

100

A+B

[minutes) 0

5 10

93.4 + 3.4 88.5 + 1 .9 0 ,2±0 .H

15 20

30

79.7+2 .9 76.8±3 .3 0 .8+0 .5

35 45 60

70 .7+2 .9

67.6+3 .0 1 .6+1 .1

65 75

66.3+3 .6

90

64.3+3 .0 2 • 3+1 .1

95 105 110

61 .4+3 .9

120

59• 9+3.3

125

100 .0

100 .0

63.8+3 .f 2 .4+1 .0

62.7+3 . 0 2 .9+1 .2

67

•

The uptake in the cal varia of t h i s hydrolyzed 99mTc i s a H O u t 20% a f t e r two hours of i n c u b a t i o n . A f a s t component was observed d u r i n g t h e f i r s t 30 m i n u t e s , f o l l o w e d by a s l o w e r component. These r e s u l t s agree with the hypothesis that 99m-pc(c;n)_MDp d i s s o c i a t e s before the uptake of ^QrT1Tc in bone. The s t a b i l i t y of the 99 m Tc(Sn)-MDP complex depends on the MDP c o n c e n t r a t i o n (13) in the medium. In vivo the MDP concentration within the bone may be too low to prevent hydrolysis. In o r d e r t o i n v e s t i g a t e i f t h e same p r o c e s s t a k e s p l a c e in t h e a b s e n c e of a r e d u c i n g agent used m t h e p r o d u c t i o n of " ! C T c l a b e l e d MD?, a 99m T c _ M D P C O mplex was formed by means of e l e c t r o l y t i c a l reduction of 9"mTc0^ in presence of excess MDP. The MD? concentration in the incubation medium i s the same as in case of the " m Te(Sn)-MD? incubation. The uptake of " m Tc-MD? i s about ao% after two hours, while the r a d i o a c t i v i t y in the incubation medium is s t i l l in the form of 99mTc-MDP a s c o n t r o l l e d with paper and gel chromatography. No hydrolysis of t h i s complex was e s t a b l i s h e d . A f a s t phase of uptake d u r i n g t h e f i r s t ^5 m i n u t e s was observed, followed by a slower phase. In e a r l i e r experiments, d i l u t i o n of 9QmTc-MDP to the same extent as " m Tc(Sn)-MDP did not r e s u l t in h y d r o l y s i s (16). C o n s i d e r i n g t h i s , t h e r e s u l t s of our study give evidence for the uptake of "^mTc-MDP as a unit. In c o n c l u s i o n : I n c u b a t i o n of f e t a l r a t c a l v a r i a in a b u f f e r e d s a l t s o l u t i o n makes i t p l a u s i b l e t h a t t h e u p t a k e of " m T e from Tc(Sn)-MDP t a k e s p l a c e a f t e r h y d r o l y s i s of t h i s complex, whereas the uptake of ^"mTc~MDP takes place as a unit.

68

References 1. 2. 3.

4.

5. 6. 7.

8.

9. 10. 11.

12.

13.

14.

Matin, P.: Appearance of bone scans following f r a c t u r e s , including immediate and long-term studies. J.Nucl.Med.20: 1227-1231, 1979 B a t i k l e s , J., Vasilas, A., P i z z i , W.F. et al.: Bone scanning in the detection of occult fractures. J.Trauma 21: 564-569, 1981 M i l l e r , S.W., Castronovo, F.P., Pendergrass, H.P. et a l . : Technetium-99m labeled diphosphonate bone scanning in Paget's disease. Am.J.Roentgenol.121: 177-183, 197^ Lisbona, R., Rosenthall, L.: Radionuclide imaging of s e p t i c j o i n t s and t h e i r d i f f e r e n t i a t i o n s form periaticular osteomyelitis and c e l l u l i t i s in pediatrics. Clin.Nucl.Med.2: 337~31*3, 1977 McNeil, B.J.: Rationale for use of bone scans in selected metastasis and primary bone tumors. Semin.Nucl.Med.8: 336-3^5, 1978 Rosenthall, L., Kaye, M.: Technetium-99m pyrophosphate kinetics and imaging in metabolic bone disease. J.Nucl.Med.16: 33-39, 1975 Garnett, E.S., Bowen, B.M., Coates, G. et a l . : An a n a l y s i s of f a c t o r s which influence the local accumulation of boneseeking radiopharmaceuticals. Invest.Rad.10: 564-568, 1975 Yano, Y., McRae, J., van Dyke, D.C. et a l . : Technetium-99mlabeled stanous ethane-1 -hydroxy-1, 1-diphosphonate: A new bone scanning agent. J.Nucl.Med.1 4: 73"78, 1973 Bisaz, S., Jung, A., Fleisch, H.: Uptake by bone of pyrophosphate, diphosphonate and their technetium derivatives. Clin.Science Mol.Med.54: 265-272, 1978 Cox, P.H.: ""mTc-complexes for skeletal scintigraphy. Physiochemical factors affecting bone and bone marrow uptake. Br.J.Rad.47: 845"850, 1974 Van Langevelde, A., Driessen, O.M.J., Pauwels, E.K.J. et a l . : Aspects of '™mTechnetium. Binding from Ethane-1-hydroxy-1, 1diphosophonate-""mTc-complex to bone. Eur.J.Nucl.Med.2: 47~51 , 1977 B i l l i n g h u r s t , M.W., J e t t e , D., Somers, E.: I n v e s t i g a t i o n of the i n t e r a c t i o n of hydroxy-apatite with ^" m Technetium in association with stannous pyrophosphate. Int.J.Appl.Radiat.Isot.32: 559~566, 1981 Schumichen, C , Korfgen, T., Hoffmann, G.: mRelationship between complex s t a b i l i t y and biokinetics of ^ Tc-phosphate compounds. Nuol.Med.19: 7-10, 1980 Francis, M.D., Ferguson, D.L., Tofe, A.J. et a l . : Comparative evaluation of three diphosphonates: In vitro adsorption (C-14 labeled) and In vivo osteogenic uptake (Tc-99m labeled). J.Nucl.Med.21: 1185-1189, 1980 69

15. Kaye, M., Silverton, S., Rosenthall, L.: Technetium-99m-pyrophosphate: Studies in vivo and in vitro. J.Nucl.Med.16: 40-45, 1975 16. Savelkoul, T.J.F., Oldenburg, S.J., van Oort, W.J. et a l . : Electrolytically labeled 99mTc_MDP: chromatographic Pattern Stability and Biodistribution in Rats. Int.J.Appl.Radiat.Isot., 1984 (in press). 17- Neuman, W.F., Brommage, R., Myers, C.R.: The measurements of Ca2+ effluxes from bone. Calcif.Tiss.Res.24: 113-117, 1977 18. Scarpace, P.J., Neuman, W.F.: The blood: bone disequilibrium I. The active accumulation of K+ into the bone extracellular fluid. Calcif.Tiss.Res.20: 137-149, 1976 19. Scarpace, P.J., Neuman, W.F.: The blood: bone disequilibrium I I . Evidence against the a c t i v e accumulation of calcium or phosphate into the bone extracellular fluid. Calcif.Tiss.Res.20: 151-158, 1976 20. Khan, R.A.A.: Factors influencing the d i s t r i b u t i o n of 99m_ Technetium methylene diphosphonate in bone and soft tissues. Experientia 34: 1598-1600, 1978

70

6. A RAPID METHOD FOR PREPARING UNDECALCIFIED SECTIONS OF BONE FOR AUTORADIOGRAPHIC INVESTIGATION WITH SHORT-LIVED RADIONUCLIDES.

T.J.F. Savelkoul, W.J. Visser, J.M.M. Roelofs, M.H.F. Lentferink.

Abstract

To prepare sections of undecalcified bone suitable both for autoradiography with short-lived radionuclides such as "9m_ Technetium (Ü, = 6 hr) and for normal histology, rapid processing is necessary. By modifying the routine technique of embedding in plastic, sections can be obtained within 6 hours. The most important modification concerns the temperature used for the different steps in the process. The procedure has been used to localize ^^ m Tc labeled methylene diphosphonate for skeletal scintigraphy.

Stain Technology 58: 1-5, 1983

71

Introduction Methylene diphosphonate labeled with 95m7echnetium (99mTc(Sn)MDP) is used in clinical studies of bone disease (1,2). Regions of elevated uptake of the radionuclide revealed by skeletal scintigraphy correspond to areas of increased bone turnover. Information about the localization of the radiotracer can be gained by studying microautoradiographs. Using plastic embedding (3.1.5), sections are of good quality, but radioactivity is almost completely lost because the processing time i s so long. Even large doses of ^^raTc(Sn)-MDP are not sufficient for autoradiography on sections prepared in this way because the "' m Tc has a h a l f - l i f e of 6 hours. Frozen sections of bone can be made rapidly but preservation of structure is inferior (6,7). In t h i s paper a rapid method of embedding is described for use with short-lived radionuclides such as ^^mTc, which s t i l l takes advantage of the excellent structural preservation afforded by plastic embedding.

Materials and Methods To t e s t the quality of the rapid method of embedding, we used a human t r a n s i l i a c a l bone biopsy specimen. For autoradiography, calvariae of f e t a l Wistar r a t s , on the 19th day of gestation, were incubated with 111 MBq (3 tnCi) "mTc(Sn)-MDP in Hanks'Balanced Salt Solution, pH 7.4, for two hours. In a second study 185 MBq (5 mCi) "mTc(Sn)-MPD dissolved in 1 ml normal s a l i n e were injected into the t a i l vein of Wistar rats two hours before they were sacrified. The femora were immediately divided longitudinally with a thin, rapidly r o t a t i n g saw, cooled with normal saline. All specimens were processed as follows: Fixation: in a buffered mixture of formalin and methanol according to Burkhardt (8); fixation time, 45 minutes. Dehydration: in absolute methanol for 1 hour; change methanol after 15 and 30 minutes. Impregnation I: in a methylmethacrylate mixture (MMA-mixture): 100 ml MMA (DA), 25 ml plastoid N (Rohm Pharma), 3.5 g a i r dried benzoyl peroxide (Merck); impregnate 1 hour; change mixture after 15 and 30 minutes. To accelerate the three steps above, fixation, dehydration and 72

impregnation I are carried out at 50°C. Impregnation I I : in preprocessed MMA-mixture at room temperat u r e , impregnation time i s 15 minutes. The preprocessing is carried out in advance by preheating the MMA-mixture for about 3 hours in a water bath at 50°C. When the MMA-mixture becomes as viscous as glycerine, the process i s stopped by cooling. Store at Polymerization: in a mold (9); the temperature in the mold is controlled by a forced c i r c u l a t i o n of water at 60°C. The r e s i n polymerizes in about two hours. Sections: cut H Mm sections with a Jung K microtome. Heat g e l a t i n coated s l i d e s to 80°C on a warming t a b l e . Mount the sections on slides. To f a c i l i t a t e flattening and adherence, put a drop of fresh gelatin solution (7 g potassium bichromate and 2 g g e l a t i n in 2.5 1 of 50? ethanol) on the s l i d e s . They w i l l be suitable both for staining and autoradiography. Autoradiography: for autoradiography use Kodak NTB2 nuclear track emulsion diluted 1:1 with doubly distilled water as recommended by the manufacturer. Dip the sections in the emulsion using a device which withdraws the coated s l i d e s from the emulsion at a low uniform speed (10). Dry the s l i d e s and place them in a l i g h t - t i g h t box sealed with tape. Expose for 18 hours at 4°C. Develop in Kodak D-170 and fix in 2^4% sodium thiosulfate. Staining: for h i s t o l o g i c a l s t a i n i n g we have used t o l u i d i n e blue, hematoxylin and eosin, and the Goldner staining.

Results Figures 1 and 2 show a transiliacal human bone biopsy and distal femur of a Wistar r a t prepared by t h i s method. The c e l l s and t i s s u e s t r u c t u r e s are as well preserved as in our routine procedure. Figure 3, an autoradiograph of a f e t a l r a t c a l v a r i a , shows clearly a preferred localization of the radioisotope at s i t e s of beginning bone formation (bone islands). Figure H shows a trabeculum in the metaphysis of a rat femur with the radioisotope localized in the mineralized bone.

73

. r..:1 - . : - 1 bone

Discussion

•^

r' '

t h e r a p : 1 •?-r.oec~.. •..• ~. >?-. r . ; : i o ^ c r ' ^ O v t --ve". : •; : : : P : a 5 e o f h-.!r..>n b o n e . Te a c ^ e l e r a t c tion,

:":xaT.i~n,

t h e terr. p e r a t j r e ^ u r : n g

:•">•".>• ::•* st i ^ri ,i:i>: t •>.> f i r s t preparation

was r-:i:sed

ir.pregna-

t o ^ 0 ° C . To

Figure 3. C a r t i l a g i n o u s p a r t of a f e t a l W i s t a l " a t c a l v a r i a ( 1 9 t h day of g e s t a t i o n ) i n c u b a t e d i n v i t r o for ? h o u r s w i t h 3 mCi " " m T c - ( S n ) - M D P . A c t i v i t y ( s e e aroows) i s l o c a l i z e d in a r e a s of new bone f o r m a t i o n . The f o l l o w i n g s t r u c t u r e s c a n be d i s t i n g u i s h e d : e c t o c r a n i a l p e r i o s t e u m (ECTP), c a r t i l a g e (C), e n d o c r a n i a l periosteum (ENDP). T o l j i l i n e o l u e . 250 X. Figure 4. Femoral shaft of a Wistar rat, 6 weeks old. Five mCi "- m Tc(Sn)-MDP were injected intravenously ? hour before sacrifice. Activity is diffusely located in trabeeular bone. Toluidine blue. 400 X.

s h o r t e n p o l y m e r i z a t i o n t i m e , t h e second i m p r e g n a t i o n i s performed in a p r e p r o c e s s e d MMA-mixture. Zare must be ta'
75

et al. (12) found a 2M hr exposure optimal in their study of cellular uptake of 99m Tc> i n Our study, we preferred 18 hours. Autoradiography of frozen bone sections using ^9mj c _ la j :)ele( j radiopharmaceuticals has been reported earlier. The radioactivity of 99mTc j n these studies was found in less mineralized and less mature bone (13). in bone matrix (1*0, adjacent to the epiphysial plate, along the Haversian canals and at the edge of trabeculae (6). Christensen and Krogsgaard (7) also found radioaotivity in Howship's lacunae on resorbing surfaces as well as in areas of new bone formation. In our study the radioactivity in the fetal rat calvaria is localized in areas of new bone formation. In the rat femoral shaft the radioactivity is localized in trabecular bone. A direct relationship to osteoblasts or osteoclasts was not observed. In conclusion, the described method of rapid embedding in MMA gives good histological pictures and makes it possible to perform microautoradiography with radionuclides with short half-lives.

76

References 1. 2.

3. 4. 5.

6. 7. 8. 9.

10. 11. 12.

13.

14.

Subramanian, G., McAfee, J.G.: A new complex of 99mTc for skeletal imaging. Radiology 99: 192-196, 1971 Subramanian, G., McAfee, J.G., B l a i r , R.J. et a l . : Technetium99m-methyl en e diphosphonate: A superior agent for s k e l e t a l imaging: comparison with other technetium complexes. J.Nucl.Med.16: 744-755, 1975 Te Velde, J., Burkhardt, R., Kleiwerda, K. et a l . : Methylmethacrylate as embedding medium in histopathology. Histopathology 1: 319-330, 1977 Spijker, H.J.D.: A procedure for obtaining thin sections of undecalcified bone biopsies embedded in methylmethacrylate. Microsc.Acta 81: 16-17, 1978 Evans, R.A., Dunstan, C.R. and Bayliuh, D.J.: Histochemical identification of o s t e o c l a s t s in undecalcified s e c t i o n s of human bone. Min.Electr.Metab.2: 179-185, 1979 Khan, R.A. Hughes, S., Lavender, P. et a l . : Autoradiography of technetium-labeled diphosphonate in rate bone. J.Bone Joint Surg.6i-B: 221-224, 1979 Christensen, S.B. and Krogsgaard, W.O.: Localization of Tc99m MDP in epiphyseal growth plates of rats. J.Nucl.Med.22: 237-245, 1981 Burkhardt, R.: Praparative Voraussetzungen zur k l i n i s c h e Histologie des menschlichen Knockenmarkes. Blut 14: 30-46, 1966 Lentferink, M.H.F., Visser, W.J., Roelofs, J.M.M. et a l . : Limitation of temperature r i s e during embedding in methylmethacrylate, using a water cooled mould. Stain Technol.55: 193-194, 1980 Kopriwa, B.M.: A semiautomatic instrument for the radioautographic coating technique. J.Histochem.Cytochem.14: 923~928, 1966 Dillman, L.L.: Radionuclide decay schemes and nuclear parameters for use in radiationdose estimation. J.Nucl.Med.10: 22, 1969 Barth, R.F., Clancy, J. and Pugh, J.M.: Autoradiographic s t u d i e s on the c e l l u l a r uptake of technetium-99m and chromium-51. J.Microsc.109: 211-222, 1976 Tilden, R.L.tf mJackson, J., Enneking, W.F., Deland, H. and McVey, J.T.: ™ Tc-polyphosphate: h i s t o l o g i c a l l o c a l i z a t i o n in human femurs by autoradiography. J.Nucl.Med.14: 576-578, 1973 Van Langevelde, A., Driessen, D.M.J., Pauwels, E.K.J. et a l . : Aspects of Technetium binding from an e t h a n e - 1 - 1 , 1diphosphonate-9"mTc complex to bone. J.Nucl.Med.2: 47-51, 1977

77

7. A MICRO-AUTORADIOGRAPHIC STUDY OF THE LOCALIZATION OF 99mTc(Sn)-MDP AND 99mTc-MDP IN UNDECALCIFIED BONE SECTIONS.

T.J.F. Savelkoul, W.J. Visser, S.J.

Oldenburg, S.A. Duursma

Abstract

The localization of ^^mTc(Sn)-MDP in bone tissue is compared with 9 9 m T c _ M D p b y m e a n s o f microautoradiography of undeoalcified bone sections. Sections of good histologic quality were obtained by a rapid embedding method in methylmethacrylate. No differences were found in the localization of both radiopharmaceuticals in fetal rat calvaria after incubation in /itro or in rat femora after administration in vivo. In the incubation experiment hydrolyzed ""mTechnetium was formed. The uptake was high in areas of new bone formation. No uptake was seen in cells or in resorbing areas. The uptake of ""mTc(Sn)-MDP in compact bone was in the vicinity of blood vessels.

Submitted for publication

78

Introduction The uptake of 9 9 m T e e h n e t i u m _ c o m p O u n d s i n b O n e j s a matter of discussion since years (1-8). Hydrolysis of 99mTc(Sn)-MDP before bone uptake i s l i k e l y (5,9). The uptake of "mTc-MDP ( e l e c t r o lytically labeled (10)) in fetal rat cal varia in a buffered salt solution take place as a unit. To evaluate the l o c a l i z a t i o n of Technetium in bone, some macro- and micro-autoradiographic studies have been performed (7-11). As the processing time for plastic embedding is long, these studies were mostly performed with frozen bone sections (5,8,12,13). In other studies wax have been used as embedding material, however in these studies 9"Tc and 95m-i-c> w ith relatively long physical half lives, were used as the tracer (13~15). The preservation of the bone s t r u c t u r e in frozen bone sections or using wax is inferior to the embedding in methylmethacrylate. Tilden et al.(17) used epoxy resin for embedding and reduced the processing time before exposure to 14 hours (2.3 "mTechnetium half lives). In a recent study we described a rapid method for preparing undecalcified sections of bone, embedded in methylmethacrylate (18). The processing time before exposure has been reduced to 6 hours (one ^^mTechnetium half life). The purpose of t h i s study is to evaluate the l o c a l i z a t i o n of "mTc(Sn)-MDP and "mTc-MDP in fetal rat calvaria in vitro and in r a t femora in vivo after administration of these radiopharmaceuticals. The localization of ^^mTc(Sn)-MDP in compact bone of a rib in a monkey is also studied.

Materials and Methods Radiopharmaceuticals 99mTc(Sn)-MDP Was prepared by dissolving 200 mg MDP (Sigma Chemical Company) and 50 mg SnClo.2H20 in 90 ml normal s a l i n e . The pH was adjusted to 7.0 by NaOH and normal saline was added to a total volume of 100 ml. One ml of this solution was mixed with 2 ml

99m

TQO~n.

99m Tc _ MDp w a s p r e p a r e c j a f t e r e l e c t r o l y t i c a l reduction of 99m TcO in t h e p r e s e n c e of MDP, as p r e v i o u s l y d e s c r i b e d (10). Before a d m i n i s t r a t i o n 99m T c _ M D p w a s s e p a r . a t e d from remaining 99m TcO by means of gel chromatography a t Biogel PH ( B i o r a d ) , 100-200 79

mesh, with normal saline as the eluant in a 0.9 x 28 cm glass column. Of both radiopharmaceuticals ascending paper chromatography was performed on Whatman 3 MM with 0.5 M a c e t a t e buffer (pH 5.0) as the eluant.

Animals Calvaria of fetal Wistar rats on the 19th day of gestation were incubated with 111 MBq (3 mCi) of either radiopharraaceutical in Hanks' Balanced Salt Solution (pH l.k, temperature 37° C) for two hours. The calvaria were clamped in modified Ussing chambers with t h e i r concave s i t e s d i r e c t e d to the compartment in which the radiopharmaceutical was administered. Male Wistar r a t s (6 weeks old) received 187 MBq (5 mCi) of either radiopharmaceutical, administered into the t a i l vein and were s a c r i f i e d two'hours l a t e r . The femora were immediately removed and divided longitudinally with a thin rapidly rotating saw, cooled with normal saline. Two Java monkeys, 30 weeks old, received 1110 MBq (30 mCi) "'mTc(Sn)-MDP intravenously. Two hours l a t e r , one of the lower ribs was resected under general anesthesia. Scintigrams were performed before the bone specimen were obtained from the rats and monkeys.

Tissue preparations and microautoradiography All bone specimen were fixated in a mixture of formaline and methanol according to Burkhardt (19) and f u r t h e r processed as previously described (18). The k \i s e c t i o n s were dipped in a nuclear track emulsion (NTB -2 Kodak), diluted 1:1 with doubly d i s t i l l e d water. The s l i d e s were exposed for 18 hours a t H°C, developed (D-170 Kodak) and fixed in 24? sodium thiosulfate. For h i s t o l o g i c a l s t a i n i n g t o l u i d i n e blue and hematoxylin/eosin was used. In all experiments unlabeled bone sections were subjected to the same procedures as a control.

80

Results The paper chrornatogramo of both radiopharmaceuti oals showed cr.ly r a d i o a c t i v i t y a t Rf 1.0 ( 9 9 m Tc(Sn)-MD?) r e s p e c t i v e l y Sf Z.'i (99m-pc_MDp) (10). However during the incubation experiment witr. 9?mTc(Sn)-MDP, reduced hydrolyzed 9 9 m T e c h n e t i j m o r i g i n a t e d 'r.f 0.0) t o an e x t e n t of 75? of a l l r a d i o a c t i v i t y . ' 5r 'T - - " I . ? remained s t a b l e in ti. s experiment. The s c i n t i g r a m s of t h e r a t s and monkeys showed a normal ocr.e uptake with l i t t l e a c t i v i t y in the soft t i s s u e s .

F i gur e 1 S e c t i o n of a f e t a l r a t c a l v a r i u m i n c u b a t e d w i t h " m T c - M D P ( T o l u i d i n e blue.MOx). R a d i o a c t i v i t y i s l o c a l i z e d d i f f u s e l y in t h e y o u n g m i n e r a l i z e d bone (mb), s u r r o u n d e d by o s t e o b l a s t s (ob). 81

F i g ur e 2. Section of a fetal rat calvarium incubated with °^mTc(Sn)-MDP (hemotoxylin/eosin, 100x). R a d i o a c t i v i t y i s especiallyl o c a l i z e d at the convex s i t e (a). At the opposite concave s i t e (b), a c t i ; e bone resorption is present and the number of osteoblasts is r e l a t i v e l y low (mb: mineralizing bone).

cc v

.£ v

-.
;

j«'

* .- ->"' «r.

£« . -

-'!*">

•-

• .

* ?

v

'-•.

'~~t

•-

• »w *?-*'* t, • '*

Sr-

f," - ,

.

F i g ur e 3 . D i s t a l f e m u r of a W i s t a r r a t , 6 w e e k s o l d , t w o h o u r s a f t e r a d m i n i s t r a t i o n of 9 9 m T c _ M D p ( t o l u i d i n e b l u e , t O x ) . R a d i o a c t i v i t y i s l o c a l i z e d m a i n l y i n t h e d e v e l o p i n g t r a b e e u l a e of t h e metaphysis ( t m ) and in t h e c a l c i f y i n g c a r t i l a g e ( c c ) .

Figure 4. Bone t r a b e c u l u m in t h e m e t a p h y s i s of t h e d i s t a l femur of a Wistar r a t , 6 weeks old, two hours a f t e r the a d m i n i s t r a t i o n of ^ " T C - M D P ( t o l u i d i n e blue, iJOx). There i s a preference of t h e developed s i l v e r g r a i n s for t h e s i t e of a c t i v e bone formation (arrows), but also uptake in the mineralized bone (mb). No uptake i s seen in the bone marrow (bm).

mb

f; bv

Figure 5. C o r t i c a l bone of a rib of a monkey, 30 weeks o l d , (" m Tc(Sn)-MDP, t o l u i d i n e b l u e , HOx). R a d i o a c t i v i t y i s localized diffusely in the mineralized bone (mb), e s p e c i a l l y in the v i c i n i t y of a blood vessel (bv).

35

In the calvariae the uptake of r a d i o a c t i v i t y fron both preparations is localized in areas of bone formation. This localization is the same for both radiopharmaceuticals (figure 1). In the growing calvariae bone resorption occurs at the concave s i t e , with bone formation at the convex s i t e . In figure 2 the radioactivity shows a preference for the s i t e of bone formation. In the rat fencra the localization of ^^mlc(Sn}-7ADP and 99n-c_ MDP is the same. Most radioactivity i s found in trabeculae below the epiphysial growth p l a t e s (figure 3). In trabecular bone, uptake in the mineralized bone is seen with a preference Tor the sites with osteoblasts (figure 4). In the ribs of the monkeys, radioactivity is localized diffusely in the mineralized bone, but more a c t i v i t y is prese r .v in the vicinity of blood vessels (figure 5). In the unlabeled sections the amount of developed silver grains is equal to the background of the labeled sections.

Discussion With the embedding in methylmethacrylate, histological sections with a good structural preservation were obtained in combination with a short processing time. The extranuclear electrons emitted by ™mTechnetium a r e o f sufficient low energy for good r e s o l u t i o n microautoradiograms. Driessen et a l . (20) found a r e s o l u t i o n of 22.6 u with a dry autoradiographic technique on s t r i p p i n g film. Barth et a l . (21) labeled Hela c e l l s and erythrocytes with "^mTechnetiurn. The exposed silver grains (NTB-3 Kodak) were randomly scattered over the entire cell area, only rarely tracks were seen radiating out from c e l l s . The r e s o l u t i o n was so good, t h a t they suggested that determination of c e l l u l a r l o c a l i z a t i o n of ""mTechnetium labeled compounds might be possible. The resolution derived from the l o c a l i z a t i o n at the erythrocytes would be l e s s than 7\x. In our experiment we used a l e s s s e n s i t i v e emulsion~(NTB-2 Kodak) diluted with doubly distilled water. Even a better resolution is expected. In e a r l i e r incubation experiments with f e t a l r a t c a l v a r i a e , uptake of "mTeohnetium after the administration of "mTc(Sn)-MDP was only seen when hydrolyzed 9"raTechnetium was formed. 99mTc-MDP however was taken up as a unit. In t h i s experiment hydrolyzed

86

"9raTechnetiiim

was formed

for

75* after

administration

of

in v i t r o . In the l o c a l i z a t i o n of developed s i l v e r grains in the microajtoradiograns however there was no difference between hydrolyzed ^^^Technetium and ^^Tc-MDP. ^ high uptake is detected in the bone germs and in the active bone forming (convex) s i t e s , with only rare grains at the resorbing (concave) s i t e s of the calvariae. The calvariae were placed in the incubations chambers with t h e i r concave s i t e s to the compartment in which the radioactivity was administered. So the radioactivity has passed the resorbing s i t e at f i r s t . The adminis t r a t i o n of 99mTc(5n)_.Jjr)p 3 n d 99mTc_./r)p j n V i v o ; n r a t s led to the same results. High uptake can be seen in the active epiphysial plates and in trabecular bone, with a preferent localization for the s i t e of active bone formation, adjacent to osteoblasts. No c e l l l a b e l i n g was detected. In compact bone diffusely s c a t tered grains in the mineralized bone are seen. Two hours after administration s t i l l radioactivity is demonstrated in the vicinity of blood vessels. In an incubation experiment with rat femora Khan et a l . (12) concluded to l o c a l i z a t i o n of the t r a c e r adjacent to the epiphysial plate and also on the surface of the bone. They found no tracer in established bone, however i t is not known if there was passage from the radiopharmaceuticals through the 5 mm bone s e c t i o n s . In our incubation with the thin f e t a l calvariae (0 3 M) we found diffuse uptake of ^^mTechnetium in the mineralized bone. The preferent l o c a l i z a t i o n for developing trabeculae of the methaphysis and at s i t e s of bone formation is described e a r l i e r (8, 12, 14). This i s in agreement with our r e s u l t s . We could not detect a preference for l o c a l i z a t i o n in resorbing a r e a s , as i s found by Christensen et a l . (8), nor in the o s t e o b l a s t s as described by Guillemart et a l . (1*0. The uptake seems to depend not only on blood supply, but also on the activity of bone formation. In conclusion: the localization of ^^mTc(Sn)-MDP does not differ from ™mTc-MDP. predominantly uptake was seen in areas of bone formation, whereas in resorbing areas no activity was detected. No cell labeling was seen. In compact bone, with relatively slow bone turnover, 9"mTc y,as detected near the blood vessels two hours after administration of "mTc(Sn)-MDP.

87

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12. 13. 14.

Yano, Y., McRae, J., van Dyke, D.C., e t a l . : Technetium 99m-labeled stannous ethane-1-hydroxy-1,1 -diphosphonate: a new bone scanning agent. J.Nucl.Med.1'4: 73-78, 1973 Cox, P.H.: 99.11x0-0011) pi exes for skeletal scintigraphy. Physicochemical factors affecting bone and bone marrow uptake. Br.J.Red.47: 845-850, 197*1 G a r n e t t , E.S., Bowen, B.M., Coates, G. et a l . : \n Analysis of f a c t o r s which influence the l o c a l accumulation of boneseeking radiopharmaceuticals. Invest.Red.10: 564-568, 1975 Kaye, M, S i l v e r t o n , S., R o s e n t h a l l , G.: Technetium-99nipyrophosphate: Studies in vivo and in vitro. J.Nucl.Med.16: 40-45, 1975 Van Langevelde, A., Driessen, O.M.J., Pauwels, E.K.J. et a l . : Aspects of Technetium. Binding from Ethane-1 -hydroxy-1 , 1diphosphonate-""mTc-complex to Bone. Eur.J.Nucl.Med.2: 47-51, 1977 F r a n c i s , M.D., Ferguson, D.L., Tofe, A.J. et a l . : Comparative evaluation of three diphosphonates: In vitro adsorption (C-14 labeled) and In vivo osteogenic uptake (Tc-99m labeled). J.Nucl.Med.21: 1185-1189, 1980 B i l l i n g h u r s t , M.W., J e t t e , D., Soraers, E.: I n v e s t i g a t i o n of the i n t e r a c t i o n of h y d r o x y - a p a t i t e with Technetium in association with stannous pyrophosphate. Int.J.Appl.Radiat.Isot.32: 559"566, 1981 C h r i s t e n s e n , S.B., K r o g s g a a r d , O.W.: L o c a l i z a t i o n of 99mTc-Methylene Diphosphonate in epiphysial Growth plates of rats. J.Nucl.Med.22: 237~245, 1981 Schumichen, C , Korfgen, T., Hoffman, G.: R e l a t i o n s h i p between complex s t a b i l i t y and biokinetics of ^^mTc-phosphate compounds. Nucl.Med.19: 7~10, 1980 Savelkoul, T.F.J., Oldenburg, S.J., van Oort W.J. et a l . : Electrolytically labeled "^mTc-MDP: Chromatographic pattern, s t a b i l i t y and biodistribution in r a t s . Int.J.Appl.Radiat.Isot: 1984 (in press) Rohlin, M., Hammerstrom, L.: Whole-body Autoradiography of 99m^c _ i a b e i e c i pyrophosphate and related compounds in young rats. Acta Rad.Ther.phys.Biol.15: 71-80, 1976 Khan, R.A., Hughes, S., Lavender, P. et a l , : Autoradiography of Technetium labeled diphosphonate in rat bone. J.Bone Joint Surg.6i-B: 221-224, 1979 Christensen, S.B., Arnoldi, C.C.: T i s t r i b u t i o n of " m T c phosphate compounds in ostearthrytic femoral heads. J.Bone Joint Surg.62-A: 90-96, 1980 Guillemart, A., Besnard, J-C., l e Pape, A. et a l . : S k e l e t a l uptake of pyrophosphate labeled with Technetium-95n) and 88

15.

16.

17.

18.

19. 20. 21.

Technetium-96, as evaluated by autoradiography. J.Nucl.Med.19: 895-899. 1978 Guillemart, A., l e Pape, A., Besnard, J-L.: Bone k i n e t i c s of Calcium-45 and pyrophosphate labeled with Technetium-96: An autoradiographic evaluation. J.Nucl.Med.21: i66-i»70, 1980 Le Pape, A., Guillemart, A.: Autoradiographic Comparison of 9°Tc-pyrophosphate and ^Ca bone uptake. Eur.J.Nucl.Med.J.7: 127-129, 1982 Tilden, R.L., Jackson, J., Enneking, W.F. et a l . : polyphosphate: Histological localization in human femurs by autoradiography. J.Nucl.Med.iH: 576-578, 1973 Savelkoul, T.J.F., Visser, W.J., Roelofs, J.M.M, et a l . : A rapid method for preparing undecalcified s e c t i o n s of bone for autoradiographic I n v e s t i g a t i o n with short-lived radionuclides. Stain.Techn.58: 1~5, 1983 Burkhardt, R.: Praparative Voraussetzungen zutn k l i n i s c h e Histologie des menschliches Knochenmarkes. Blut U: 30-46, 1966 Driessen, D.M.J., Thesingh, C.W., v.d. Bosch, N.: Autoradiographic model experiments with 'Ga and ™"mTc. Histochemistry 50: 77"8O, 1976, Barth, R.F., Clancy J., Pugh, J.M.: Autoradiographic s t u d i e s on the cellular uptake of Technetium-99m and Chromium-51. J.Microsc.109: 211-222, 1976

89

8. GENERAL DISCUSSION AND SUMMARY

Radionuclide bone imaging has been demonstrated to be a sensitive means for the detection of early osseous disease. Focally increased uptake of bone-seeking radiopharmaceutieals arises in areas of increased metabolic activity. The blood flow is generally higher in these areas, but t h i s i s not the only reason for an increased uptake. If blood flow i s disturbed, there will be no uptake in combination with decreased metabolic a c t i v i t y , and osteonecrosis can develope. If only osteolysis exists, with no osteogenic respons, fotopenic lesions will be detected at the scintigram. As soon as osteogenesis appears, the scintigram will reverse and the formerly "cold" lesions become "hot". The uptake takes place in mineralizing areas and i s in fact dependent on osteoblastic activity. So the scintigram can be seen as a derived reflection of osteoblastic activity. Then i t must be possible to quantitate in one way or another this activity, especially if the whole skeleton i s involved. Some investigators have performed uptake s t u d i e s , but they are not routinely used, because of the variability in results or the complicated procedure. A problem in this evaluation is that the uptake of ^^mTc(Sn)-MDP is not clearly defined. In an electrolysis procedure without the presence of contaminating reductants a "*mTc-MDP complex is formed with clear bone-seeking properties. The scans performed in experimental animals are comparable in quality with ""mTc(Sn)-MDP scans. The chromatographic properties established on paper and gel chromatography are s l i g h t l y different. However, if higher MDP concent r a t i o n s are used in the e l e c t r o l y s i s procedure, at l e a s t two "S>mTc-MDP complexes can be formed. During the incubation with Human Serum Albumine (HSA) a conversion to one complex appears to arise. This will probably also happen in vivo and is in agreement with the finding, thai; only one complex i s excreted in urine. However, further analysis of the ^"mTc-MDP complex is needed. The complex is more stable to dilution, but in vivo some pertechnetate can arise. ^^mTC-MDP and ^^mTc(Sn)-HDP are excreted in urine with maintenance of their bone-seeking properties. In the experiments with 99mTc-MDP n 0 reduced hydrolyzed 99m T e c l l n e t i u m i s d e _ tected. In the incubation experiment of fetal rat calvaria in a buffered

90

s a l t solution, the uptake of 99mTc_MDp w a s clearly higher than the uptake of hydrolyzed 99m T e c n n e t i u r n > N o u p t a ke of 99mTc(Sn)_ MDP could be demonstrated. This i s a d e f i n i t e indication that 99mTc(Sn)-MDP i s not taken up as a unit, but that hydrolysis is necessary before uptake can take place. Some hydrolysis i s already demonstrated in the incubation experiment with HSA, but probably most hydrolysis occurs in the e x t r a c e l l u l a r fluid m spaces. In the incubation experiment all Tc-compounds passed m through the calvaria, but only 99 Tc-MDP an( j reduced hydrolyzed 99mTechnetium were taken up by the calvaria. For the autoradiographic evaluation undecalcified bone sections embedded in methylmethacrylate were used. With this technique of p l a s t i c embedding sections of good h i s t o l o g i c quality are obtained. The problem with short-lived radionuclides, such as ""mTechnetium, is that radioactivity is almost completely lost during the long processing time. By modifying our routine procedure i t was possible to reduce the processing time to one half l i f e time of "™mTechnetium. With this rapid method sections of comparable quality with the routine procedure were obtained. The localization of ^^mTechnetium after administration of ^^mTc-MDP and ™mTc(Sn)-MDP i n v i v o atl(j i n vitro gave the same results. In vitro again reduced hydrolyzed ^^mTechnetium existed. "™mTechnetium was mainly located in areas of bone formation. In resorbing areas no activity was detected. In compact bone with relatively slow bone turnover, "*mTechnetium w a s located in the vicinity of blood vessels. Cell labeling was not demonstrated. The autoradiographic r e s u l t s were in agreement with the in vivo data of increased uptake in areas of osteogenesis. As ""mTc-MDP is taken up as a unit, t h i s may be a better agent to evaluate the osteoblastic activity in the skeleton and to quantitate the activity in uptake studies. Further research has to be performed to establish the possibilities of this radiopharmaceutical. With respect to the studied items, the following conclusions can be made: 1. I t is possible to prepare e l e c t r o l y t i c a l l y a ""mTc-MDP complex avoiding contamination with any reducing agent. Tin is not necessary for bone uptake. In comparison with ^^mTc(Sn)-MDP, the paper'and gel chromatographic properties differ slightly. The stability of 99mTc-MDP to dilution in v i t r o i s higher. The bone-seeking properties

91

are comparable. 2. The HSA-binding of 99inTc-MDP is slightly less than 99mTc(Sn)MDP. Both complexes are excreted in the urine with maintenance of their bone-seeking properties. 3. The uptake of "mTc-MDP i s f a s t e r and higher than the uptake of reduced hydrolyzed 99mTechnetium. Uptake of "mTc(Sn)-MDP in bone can only take place a f t e r decomposition of the complex. i*. With the development of a rapid embedding method for undecalcified bone sections, i t is possible to perform microautoradiography. Radioactivity i s located in areas of bone formation. No difference in l o c a l i z a t i o n of "mTc-MDP and 99m Tc(Sn)-MDP (hydrolyzed 99mTc) can be established.

92

9. SAMENVATTING Skeletscintigrafie i s een gevoelige methode voor het aantonen van botafwijkingen in een vroeg stadium. Op plaatsen met een verhoogde metabole a k t i v i t e i t worden de botzoekende radiopharmaca in verhoogde mate gestapeld. De bloedtoevoer i s over het algemeen ook toegenomen in deze gebieden, maar dit i s niet de enige reden voor de verhoogde mate van stapeling. Indien de bloedtoevoer is onderbroken, zal echter geen a k t i v i t e i t worden aangevoerd. Er i s dan tevens een verminderde metabole a k t i v i t e i t en kan botnecrose ontstaan. Botafbraak, niet gevolgd door nieuwe botaanmaak, result e e r t in het t e r plaatse ontbreken van r a d i o a k t i v i t e i t op het scintigram ("koude haard"). Zodra botnieuwvorming optreedt, zal in verhoogde mate r a d i o a k t i v i t e i t worden gestapeld en een vroegere "koude haard" zal "heet" worden. De opname vindt plaats in gebieden waar sprake i s van (nieuwe) mineral i s a t i e en i s in feite afhankelijk van de aktiviteit van osteoblasten. Het scintigram kan dan ook gezien worden als een afspiegeling van deze osteoblastische aktiviteit. Het zou mogelijk moeten zijn om deze aktiviteit op een of andere manier te kwantificeren, zeker indien het gehele skelet in een ziekteproces is betrokken, zoals bij metabole botziekten. Enkele onderzoekers hebben opname studies ontwikkeld, maar deze hebben geen algemene navolging gekregen vanwege de variabele resultaten of vanwege de gecompliceerde procedure. Een probleem i s , dat over de opname van ^"mTc(Sn)-MDP onvoldoende duidelijkheid bestaat. Via e l e c t r o l y s e zonder de aanwezigheid van contaminerende reductiemiddelen is een ^^mTc-MDP complex verkregen met duidelijke botzoekende eigenschappen. De seintigrammen, verkregen bij proefdieren, zijn vergelijkbaar in kwaliteit met de ^^mTc(Sn)-MDP scintigrammen. De chromatografische eigenschappen (papier en gel chromatografie) verschillen slechts weinig. Bij hogere MDP concentraties in het electrolysaat kunnen tenminste twee ^^mTc-MDP complexen worden gevormd. Tijdens incubatie met Humaan.Serum Albumine (HSA) ontstond er een complex. Het is waarschijn dat dit in vivo eveneens gebeurd en komt overeen met de bevinding, dat ook maar een complex wordt uitgescheiden in de urine. Verdere analyse van d i t Wm"Tc-MDP complex i s echter noodzakelijk. Dit complex i s s t a b i e l e r bij verdunning, maar in vivo kan wat per-

93

technetaat ontstaan. 99mTc_MDp en "raTc(Sn)-MDP behouden hun botzoekende eigenschappen na uitscheiding met de urine. In de experimenten met 99DITC_MDP werd geen gereduceerd gehydrolyseerd 99"1Technetium aangetoond. In een incubatie experiment van foetale skeletdakjes van ratten b l i j k t , dat de opname van 99mTc_MDp n o g e r i S dan de opname van gehydrolyseerd 9 9 m T e c h n e t i u m > Opname van 99mTc(Sn)_MI)p ^ s n j e t geconstateerd. Hieruit valt af te leiden, dat 99mTc(Sn)_MDp n i e t a l s i n t a c t complex wordt opgenomen door bot, maar dat e e r s t ontleding nodig i s , waarbij het gehydrolyseerd " 9 m T e c ] i n e t ^ u m apart wordt genomen. Deze hydrolyse vindt waarschijnlijk voor het grootste deel p l a a t s in de e x t r a c e l l u l a i r e vloeistof. Alle gebruikte 99mTc-verbindingen passeren de schedeldakjes, maar alleen 99mTc_MDp e n gereduceerd gehydrolyseerd ^^mTechnetium worden opgenomen. Om autoradiografisch de l o c a l i s a t i e van 9 9 m T e e n n e t i u m t e evalueren in niet ontkalkte botcoupes, ingebed in methylmethacrylaat, was het noodzakelijk de bewerkingsprocedure in tijd te beperken, gezien de korte halfwaarde tijd van ^^mTechnetium. Het was mogelijk deze t i j d te beperken t o t een halfwaarde t i j d (6 uur). De verkregen coupes zijn vergelijkbaar in kwaliteit met de routinematige vervaardigde coupes. Er is geen verschil in localisatie van "mTechnetium na toediening van "mTc-MDP of 99mTc(Sn)MDP in vivo in r a t t e n en in v i t r o na incubatie van foetale schedeldakjes. ^9mTechnetium is hoofdzakelijk gelegen in gebieden met botaanmaak. In gebieden met botafbraak wordt geen r a d i o a k t i v i t e i t gedetecteerd. In compact bot met een r e l a t i e f lage botombouw bevindt het ""mTechnetium zich 2 uur na injectie nog steeds in de nabijheid van bloedvaten. Label ing van cellen wordt niet gezien. Aangezien ""mTc-MDP als een geheel wordt opgenomen, l i j k t dit een beter radiopharmacon om de osteoblastische a k t i v i t e i t in het skelet te beoordelen en om de radioaktiviteit te kwantificeren door middel van opname s t u d i e s . Meer onderzoek naar de mogelijkheden van dit radiopharmacon zal hiervoor nodig zijn. De volgende konklusies komen uit deze studie naar vorens 1. Het is mogelijk om electrolytisch een 99mTc_^Dp c o m p i e x te vervaardigen zonder potentiële verontreiniging door een reducerend agens. Tin is niet noodzakelijk voor de opname van 99mTechnetium in bot. In vergelijking met 99mTc(Sn)_MDp v e r _

s c h i l l e n de eigenschappen op papier en gel chromatografie slechts weinig. De s t a b i l i t e i t van 99mTcrMDp b i j verdunning in v i t r o i s hoger. De botzoekende eigenschappen zijn vergelijkbaar. De binding aan HSA is voor 99mTc_MDp i e t s m i n c i e r uitgesproken dan voor 99mTc(sn)-MDP. Beide complexen worden uitgescheiden in de urine met behoud van hun botzoekende eigenschappen. De opname in v i t r o van ^9mTc_^Dp ^ s s n e l l e r en hoger dan de opname van gereduceerd gehydrolyseerd "mTechnetium. Opname van 99m>rc(gn)_M0p V i n ( jt alleen p l a a t s na ontleding van het complex. Met de ontwikkeling van een s n e l l e inbed methode voor n i e t ontkalkte botcoupes i s het mogelijk om micro-autoradiografische studies te verrichten. De radioaktiviteit is gelocal i s e e r d in gebieden met botaanmaak. Er i s geen verschil in localisatie voor de verschillende gebruikte preparaten.

95

CURRICULUM VITAE

De schrijver van dit proefschrift werd geboren op 30 december 1917 te Grevenbicht (L). De HBS-B opleiding werd gevolgd aan het Bisschoppelijk College St. Jozef t e S i t t a r d . Hij studeerde Geneeskunde aan de R i j k s u n i v e r s i t e i t te Utrecht van 1967 t o t 1975. Tijdens de wachttijd voor de co-assistentschappen, gedurende het studiejaar 1972-1973, was hij leraar Biologie aan de HAVO-top van de pedagogische Academie Mariahoeve te fs-Gravenhage. Na het artsexamen werd de uitgebreide cursus Nucleaire Geneeskunde van het Ministerie van Onderwijs en Wetenschappen gevolgd (september 1975 tot maart 1976). Tijdens deze cursus werd het examen Stralingshygiëne C met goed gevolg afgelegd. De stage van 3 maanden werd gevolgd in het I n s t i t u u t voor N u c l e a i r e Geneeskunde van het Academisch Ziekenhuis Utrecht (Hoofd: prof.dr. K.H.Ephraim). Na deze stage tot 1 januari 1977 arts-assistent op deze afdeling. Vanaf 1 januari 1977 t o t 1 januari 1981 in opleiding tot internist op de afdeling Inwendige Geneeskunde van het Academisch Ziekenhuis Utrecht (Hoofden: prof.dr. J.van der Sluys Veer en prof.dr. A.Struyvenberg). Inschrijving in het Specialistenregister als internist volgde op 1 januari 1981. Per 1 januari 1978 inschrijving als Nucleair Geneeskundig deels p e c i a l i s t in het Register van de Nederlandse Vereniging voor Nucleaire Geneeskunde. Vanaf 1 januari 1981 a l s wetenschappelijk hoofdambtenaar in d i e n s t van het R i j k s i n s t i t u u t voor Volksgezondheid en Milieuhygiëne, unit Nationaal Vergiftigingen Informatie Centrum (Hoofd: p r o f . d r . A.N.P. van H e i j s t ) , a f d e l i n g Medische Toxicologie (Hoofd: dr. B.Sangster). Tevens maakt hij sinds die tijd deel uit van de staf van de afdeling Interne Geneeskunde en de a f d e l i n g Reanimatie en k l i n i s c h e Toxicologie van het Academisch Ziekenhuis te Utrecht.

96