Evaluation of a DMSA kit for instant preparation of 99mTc(V)—DMSA for tumour and metastasis scintigraphy

NW/. Med. Biol. Vol. 19, No. 8, pp. 825-830, 1992 Inl. J. Radiat. Appl.Instrum.Part B Printed in Great Britain. All rights reserved

0883-2897/92 SS.00 + 0.00 Copyright 0 1992 Pcrpmon Press Ltd

Evaluation of a DMSA Kit for Instant Preparation of ggm Tc(V)--DMSA for Tumour and Metastasis Scintigraphy U. P. S. CHAUHAN,

A. BABBAR,

R. KASHYAP

and R. PRAKASH

Institute of Nuclear Medicine and Allied Sciences, Lucknow Marg, Delhi-110054, India (Received 2 April 1992) A kit has been developed to instantly prepare 99”Tc(V)-DMSA. The freeze-dried kit consisting of DMSA, stannous chloride and ascorbic acid in appropriate proportions, produces quality WmTc(VjDMSA when mixed with 0.2 mL of 3.5% NaHCO, solution and 2-4 mL of [*Tc] pertechnetate. The radiopharmaceutical characterized by chromatography with ITLC-SG in 0.9% saline and horizontal paper electrophoresis in 50 mM vemol buffer, pH 8.6, at a potential gradient of 15 V/cm showed a different mobility with respect to PPmT~(III)-DMSA, a known agent for kidney imaging. The new agent exhibited less plasma protein binding as compared to that of 99”Tc(III)-DMSA. Biodistribution of the pentavalent DMSA in mouse demonstrated greater uptake in bone and muscle and lower uptake in liver and kidney with respect to trivalent DMSA. The soft tissue tumour specificity and its suitability for tumour scintigraphy was apparent from the scintigrams of mammary carcinoma in a C, H Jax mouse and medullary carcinoma in a patient. Brain metastatic lesions were also visible in a breast carcinoma patient after administering him with the agent.

Introduction 99”Tc-dimercaptosuccinic acid (DMSA) is considered an excellent kidney imaging agent and provides quantitative information on renal function (Lin et al., 1974; Tayler et al., 1980). However, there is a lack of agreement concerning the nature of the complex (Handmaker et al., 1975; Arnold et al., 1975). A very promising tumour-specific %TcDMSA differing drastically in biodistribution from the earlier one has been reported (Ohta et al., 1984, 1985; Yokoyama et al., 1985). Though the structural characteristics of the two species of *Tc-DMSA could not be ascertained, the kidney-specific agent has been suggested to be incorporated with technetium in the valency state of either 4 or 3, whereas in the tumour-specific species, technetium is claimed to be in the 5 valency state (Hata et al., 1983). This difference in the valency states of technetium is presumably due to the difference in the pH of the two reactions. Unlike the kidney-specific agent, which is prepared at pH 2-3, the tumour-specific agent is prepared at pH 8.0-8.5. Since the kit for the preparation of the tumour-specific *Tc-DMSA is not available commercially, we have developed and evaluated it with respect to normal tissues in an animal model as well as in cancer patients.

Materials and Methods Preparation of * Tc(V)-DMSA

kit

It was prepared from a DMSA kit developed by us (Babbar et al., 1991). In brief, to 1 mL of water, NMB

WS-B

1.l mg of DMSA and 1.26 mg of NaHCOS were mixed until a clear solution was obtained. To the above solution, 0.75 mg of ascorbic acid and 0.2 mg of SnCl, were dissolved and the final pH of the solution was adjusted to 2.5 by 1 N HCl. The preparation was filtered through a 220 nm Millipore fllter, lyophilized and sealed under nitrogen atmosphere. The kits were stored at 47°C until used up to a period of 3 months. At the time of use, an aliquot of 0.2 mL of 3.5% NaHCO, was mixed with the above kit to raise its pH to 8.5, followed by adding 2-3 mL of [99mTckpertechnetate solution of requisite radioactivity (5-20mCi). For the preparation of *Tc (III)-DMSA, $‘“Tc]pertechnetate was added to the kit without adding any NaHCO,. Characterization

%Tc(V)-DMSA was characterized by chromatog raphy on Whatman No. 3 using 100% acetone, ITLC-SG and Biogel P- 100 column (1 x 20 cm) using 0.9% NaCl and paper electrophoresis in vemol buffer (50 mM) at pH 8.6. Plasma protein binding was determined in vitro as well as in uivo. The agent was incubated with 10~01 of plasma for 30min at 37°C or administered iv. to a rabbit and its blood was withdrawn at different time intervals. The protein from plasma was precipitated by adding equal volumes of 12.5% trichloroacetic acid. The supematant was removed by centrifugation and the radioactivity was measured in the protein pellet to account for protein bound radioactivity. Various factors, such as 825

826

U. P. S. CHAUHAN et al.

effect of pH, ascorbic acid and stannous chloride concentrations and storing conditions which influence the quality of *Tc(V)-DMSA preparation, have been dealt with elsewhere (Babbar et al., 1991). Animal experimentation

Blood clearance of the radiopharmaceuticals was studied in rabbits, whereas the tissue distribution study was carried out in normal mice after administering 100 ,uCi of the pentavalent and trivalent agents i.v., respectively, to each rabbit and 1 &i to each mouse. Tumour specificity of the pentavalent agent was evaluated in mammary carcinoma-bearing mice (CXH Jax). The animals were administered i.p. with 10 PCi of the radiopharmaceutical and scintigraphs were taken in a y-camera at 3 h post-injection.

Time

(min)

Fig. I. Blood clearance of 99”Tc(IIIkDMSA (A A) and *Tc(V)-DMSA (0 0) in rabbit. The radiopharmaceuticals were i.v. administered (100 PCi) and the blood was withdrawn at different time intervals. Radioactivity expressed as percent dose injected. Values are shown of 2 separate experiments.

Evaluation in cancer patients

at 4 h after their administration to rabbits. No further degradation of the radiopharmaceuticals was subsequently noticed even at 24 h. ITLC-SG chromatography in 0.9% NaCl and electrophoretic mobilities of 99mT~(IIIbDMSA and 99mTc(V)--DMSA in 50 mM vemol buffer, pH 8.6, at a potential gradient of 15 V/ cm had demonstrated different chromatographic and Results and Discussion electrophoretic mobilities of the two DMSA prepRadiochemical purity and stability of *Tc(V)arations, augmenting the contention that tumourDMSA and 99mTc(III~DMSA in vitro and in vivo in seeking 99mT~(VbDMSA differs significantly in the blood after administering to rabbits were studied physico-chemical properties from WmT~(IIIt up to a period of 24 h. The samples were subjected to DMSA. The Rf value of the pentavalent DMSA by paper chromatography using acetone and ITLC ITLC was 0.6, whereas that of the trivalent DMSA using 0.9% saline as mobile phase. Data clearly was 0.0. demonstrated that the yield of 99”Tc-labelled trivalent That the two species of %“‘Tc-DMSA differ signifiand pentavalent DMSA was more than 99% and cantly in plasma protein binding is evident from the both species were stable up to 24 h in vitro. Absence data in Table 1. Binding of WmTc-DMSA to plasma of any significant amount of reduced colloidal %Tc proteins was only 60% in vitro, whereas it was 98% was ruled out since no measurable amount of radioin the case of 9emTc(III)-DMSA. More or less similar activity was detected when ITLC was performed binding of the two agents was obtained when the which allowed the movement of pertechnetate and preparations were administered to rabbits and the *Tc(V)-DMSA leaving the reduced colloidal 99mT~ blood was withdrawn at different time intervals. It at the base. To rule out further the absence of reduced may be further inferred from the data that the colloidal *Tc, *“Tc(V jDMSA was also subjected proportion of plasma protein binding does not to Biogel P-100 column (1.2 x 20 cm) chromatogchange in vivo up to 3 h irrespective of the type of raphy. A single peak at 20 mL, far from its void DMSA preparation. Although TCA precipitation for volume where a reduced colloidal *Tc peak was determining protein bound activity does not discrimilikely, indicated the absence of reduced colloidal nate between colloidal and protein bound activity, 99mTc. Though no appreciable degradation of both significantly less in vivo plasma protein binding of the the preparations in vitro was observed up to a period *Tc(V)-DMSA (66%) as compared to that of of 24 h, about 5% degradation of trivalent DMSA 99mTc(III)-DMSA (98%) cannot be considered an and 7% of pentavalent DMSA was perceptible even artifact, especially since the preparation was free from colloidal activity. The net activity in the blood Table 1. Protein binding of *Tc (III)-DMSA and *Tc of pentavalent DMSA-treated rabbits was much less (V)-DMSA. Data are expressed as percent radioactivity bound to compared to that of trivalent DMSA-treated rabbits proteins. Each value is the mean of 3 separate experiments throughout the experimental period (Fig. 1) which RmTc (III)-DMSA *Tc (V)-DMSA Duration could be appreciated due to relatively less binding of (min) In viva In uirro In uivo In vitro this radiotracer to the plasma proteins. However, the IS 96 66 rate of trivalent DMSA clearance from the blood was 30 97 98 63 60 faster with respect to pentavalent DMSA. Further60 96 62 120 96 60 more, no appreciable radioactivity was found to be 180 9s 60 associated with the blood cells (data not shown).

Patients suspected of neoplasm with primary and secondary lesions were i.v. administered with and were scinti5-20 mCi of *Tc(V)-DMSA graphed thereafter at l-3 h in a y-camera to locate the malignant lesions.

Fig. 2. Posterior scintigram of a mammary carcinoma-bearing C,H Jax mouse. The tumour is clearly seen on the right thigh. The imaging was done at 3 h after i.p. administration of 10 PCi of 99mTc(VtDMSA.

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Fig. 3. (A) [WmTc]Pertechnetate scan of a patient, suspected of medullary carcinoma of the thyroid, taken of 5 mCi of the agent. The cold spot on the left lobe suggests the 15 min after i.v. administration malignancy site. (B) *“Tc(V jDMSA scan of the same patient 3 h after administration of 10 mCi of the radiopharmaceutical. Manifold accumulation of the agent at the malignancy site can be clearly seen which was further confirmed by biopsy.

828

%Tc(V)-DMSA

for tumour scintigraphy

Table 2. Biodistribution of %Tc (III)-DMSA and %Tc (Vt_ DMSA in mice. Each value is the mean (SE) of 5 animals

% Radioactivity per whole organ/tissue %‘I’c (III)-DMSA Organ Blood’ Liver Kidneys Intestines Bone Muscle

lh

3h

1.75 (0.18) 12.72 (1.61) 6.35 (0.31) 0.52 (0.06) 4.58 (0.43) 3.43 (0.30)

I .03 (0.11) 13.78 (0.26) 5.43 (0.16) 0.33 (0.12) 4.67 (0.38) 2.97 (0.42)

%Tc (V)-DMSA lh

3h

1.95 (0.18) 1.20 (0.07) 1.24 (0.06) 0.36 (0.06) 18.58 (1.20) 7.10 (0.67)

0.75 (0.05) 0.95 (0.04) 0.80 (0.03) 0.24 (0.08) 15.32 (0.78) 6.48 (0.86)

*The whole body blood, bones and muscles are taken as 7, 10 and 40% respectively, of the total body weight.

Biodistribution of pentavalent DMSA in mice is compared with that of trivalent DMSA (Table 2). Liver, kidneys, bones and muscle showed a significant difference in the distribution of the two DMSA preparations, whereas no such difference was seen in the case of the intestines. In the case of wmTc(V)DMSA, the radioactivity in the liver and kidneys was much less with respect to that in the case of 99mTc(III)-DMSA. However, this was the opposite to that in the bones and muscles. The Japanese group, who were the first to develop *Tc(V)-DMSA, also reported greater uptake of this agent in bone and muscle and less in liver and kidneys as compared to that of 99”Tc(III)-DMSA (Yokoyama et al., 1985). Similar observations of high bone uptake of 99mTc(V)--DMSA in rabbits has been recently reported (Watkinson et al., 1991) inferring its in vivo behaviour like a bone scanning agent. Although it is curious as to what causes the different tissue specificity of the two *Tc-DMSA preparations, no satisfactory explanation is available at present. However, others have not observed any bone specificity of the pentavalent DMSA over the trivalent DMSA (Jeghers et al., 1987; Ramamoorthy et al., 1987), not because its bone uptake is less but essentially because of the high bone uptake of the trivalent DMSA (Ramamoorthy et al., 1987). It is, therefore, certain that the kit developed by us produces %Tc(Vk DMSA similar to that of the Japanese group (Ohta et al., 1985; Yokoyama et al., 1985; Hata et al., 1983) whereas preparations of others probably differ in composition. Subsequently, pentavalent DMSA was evaluated in C3H Jax mice bearing mammary carcinoma. The specificity for this carcinoma is evident from the scintigram of the animal (Fig. 2). Finally, clinical evaluation of the agent for the scintigraphy of a variety of tumours and their metastasis in various organs clearly demonstrated its suitability as a radiopharmaceutical of choice not only for the scintigraphy of soft tissue tumours but also for their metastasis in bones, brain and liver (Kashyap et al., 1992). Selective

uptake

of the agent

in medullary

829

carcinoma of the thyroid of a patient is one such example indicating high specificity of %Tc(V)DMSA for the tumour (Fig. 3). The same location of the tumour in the patient was perceptible as a cold spot when [99mTc]pertechnetate was administered to the patient, which validates the suitability of the agent for scintigraphy of this type of tumour. Suitability of *Tc(VtDMSA for the detection of bone metastasis was ascertained in a number of cancer patients already confirmed for bone metastasis by %Tc-MDP (Kashyap et al., 1992). The results were in agreement with those of 99mTc-MDP and are in confirmation with earlier reports (Jeghers et al., 1987). These findings extend the application of 99mTc(V)-DMSA for bone metastasis detection. Whether the pentavalent DMSA is also suitable for the detection of brain metastasis has already been ascertained by examining brain metastasis in breast cancer patients after administering them with the above agent. Detection of brain metastatic lesions in a breast carcinoma patient documented, for the first time (Babbar et al., 1991), the potential of pentavalent DMSA for brain metastasis scintigraphy. Acknowledgements-The authors are grateful to Professor Viney Jain, Scientist ‘G’ and Dr (Maj. Gen.) M. L. Sapra, AVSM, VSM, Director of the Institute for their kind interest in this study.

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