ORIGINAL ARTICLE 

Annals of Nuclear Medicine Vol. 10, No. 4, 419-423, 1996 

Evaluation of Tc-99m(V) DMSA for imaging inflammatory lesions: An experimental study 

Meral T. ERCAN,* Nedim C.M. GOLALDI,* ISll S. UNSAL,* Mehmet AYDIN,* irfan PEKSOY* and Zafer HASCELiK** 

Departments of *Nuclear Medicine and **Physical Medicine and Rehabilitation. Faculty of Medicine, Hacettepe University, Ankara. Turkey 

The present study evaluated 99m-Tc(V) DMSA as an agent for the visualization of inflammatory lesions in comparison to 99m-Tc(III) DMSA and 99m-Tc-HIG. All three radiopharmaceuticals were prepared with commercial kits. 99m-Tc(V) DMSA was prepared at neutral pH by the addition of first bicarbonate and then pertechnetate to the kit contents. The labeling efficiency was 99% as determined by ITLC. Abscesses were induced by i.m. injection of 50 ul turpentine into the right thighs of 36 Swiss albino mice. Six days later 3.7 MBq of each radiopharmaceutical was i.v. administered to 12 mice. The mice were sacrificed at 1, 3, 6 and 24 h later. Scintigrams were obtained with a gamma camera. The abscesses were better visualized on scintigrams with 99m-Tc(V) DMSA compared to 99m-Tc(III) DMSA, starting at 1 h. The animals were dissected and the organs were removed, weighed and the radioactivity determined with a gamma counter. The abscess to other tissue ratios were higher with 99m-Tc(V) DMSA than the other radiopharmaceuticals. The max. abscess/muscle ratios were 9.46 +- 3.20 (24 h), 4.19 +- 1.39 (6 h) and 5.98 +- 1.17 (24 h) and max. abscess/blood ratios were 6.22 +- 1.41 , 4.09 +- 0.84 and 0.914 +- 0.351 all at 24 h for 99m-Tc(V) DMSA, 99m-Tc(III) DMSA and 99m-Tc-HIG, respectively. 
 Experimental arthritis was produced in 6 New Zealand white rabbits by intra-articular injection of ovalbumin. Four days later 37 MBq of 99m-Tc(V) DMSA and 99m-Tc-HIG were each i.v. admini-stered to 3 rabbits. Scintigrams obtained at l, 3, 6, and 24 h clearly demonstrated arthritic joints. ROI's over arthritic joints were compared to contralateral normal joints (A/C). The max. A/C ratios were 2.10 +- 0.31 (3 h) and 2.92 +- 0.99 (24 h) for 99m-Tc(V) DMSA and 99m-Tc-HIG, respectively. 
 Our results indicated the feasibility of imaging inflammatory lesions with 99m-Tc(V) DMSA. 

Key words: 99m-Tc(V) DMSA, 99m-Tc(III) DMSA, 99m-Tc-HIG, inflammation, arthritis 

INTRODUCTION 

THE ACCUMULATION of 99m-Tc(V) DMSA (dimercaptosuccinic acid) by tumoral tissues has been well documented by both experimental l-3 and clinical studies.4-7 The chemical identity of the pentavalent state was determined by analytical methods 1,8 and its efficacy in imaging soft tissue tumors was compared with the commonly used renal agent 99m-Tc(III) DMSA both in mice l and humans.9 The biodistribution in mice showed faster blood clearance and higher bone and muscle uptake with 99m-Tc(V) DMSA compared to 99m-Tc(III) DMSA.1 In man tumors and metastases were well delineated with Tc in the pentavalent but not in the trivalent state,9 although the ligand was the same. Tumor affinity of 99m-Tc(V) DMSA was attributed to the structural similarity of TcO4E-3 core to PO4E-3ion.4 
 99m-Tc(V) DMSA is a nonspecific agent and as such it is expected to localize in inflammatory lesions like other small molecular weight coordination complexes of Tc mainly by a common mechanism of infiltration into the interstitial space due to increased capillary permeability.10 Uptake of 99m-Tc(V) DMSA by inflammatory tissues has been reported by several groups,4,5,7,ll but not fully investigated. It has the advantages of an ideal 99mTc label, simple preparation method from commercially available DMSA kits and being inexpensive. It is a candidate for wide usage as an agent for imaging inflammation in clinical application if the efficacy is demonstrated. In the present study the feasibility of scintigraphic imaging of experimental abscesses and arthritis was investigated in a comparative study with 99m-Tc(V) DMSA, 99m-Tc(III) DMSA and 99m-Tc-HIG (human immuno gamma globulin) in the hope of subsequent clinical application. 

MATERIALS AND METHODS 

Radiopharmaceuticals 
All three radiopharmaceuticals (RPs) were prepared with commercial kits. Kits for preparing 99m-Tc(III) DMSA were purchased from Sorin Biomedica S.p.a., Italy. The technetium generators and HIG kits were obtained from Mallincrodt Medical B.V., Holland. 99m-Tc(III) DMSA and 99m-Tc-HIG were prepared by the addition of appropriate amounts of 99m-Tc-pertechnetate to the kit vials according to the instructions supplied by the manufacturers. 99m-Tc(V) DMSA was prepared according to the previously published procedure: 12,13 O.12 ml sterile 7% solution of NaHCO3 was added to the lypophylized kit material. When the solid material was dissolved completely 740 MBq 99m-Tc-pertechnetate in 1-3 ml was injected into the vial and the mixture was incubated at room temperature for 10 minutes. All three RPs were analyzed by impreg-nated-thin-layer-chromatography (ITLC) with ITLC-SG mini-strips and methyl ethyl keton or saline as solvents. The labeling efficiency was 99% for all three RPs. 

Animal studies 
The animal studies were carried out in accordance with the British animal protection practice (UFAW Handbook, 2nd Edn). 50 u1 turpentine was injected into the right thigh muscle of each of 36 Swiss albino mice. They were divided into 3 groups of 12 mice. Six days later the mice were injected through the tail vein with 3.7 MBq in O.1 ml of 99m-Tc(V) DMSA, 99m-Tc(III) DMSA or 99m-Tc-HIG. They were sacrificed in groups of 3 at l,3,6, and 24 h. Static images of the mice were obtained with a gamma camera (Toshiba GCA 601 E), fitted with a LEAP collimator. The mice were dissected. All the organs, the abscess, some muscle tissue from the contralateral leg, and blood and urine samples were obtained, weighed and counted in a gamma counter (Berthold, BF 5300, Germany) against a standard prepared from l/lOO dilution of the injected solution. % uptake/g tissues, and abscess/liver (A/L), blood (A/B), muscle (A/M), intestine (A/1) and kidney (A/ K) ratios were calculated. 

 Experimental arthritis was produced in 6 New Zealand white rabbits (3.0-3.5 kg) by intra-articular injection of l ml ovalbumin (Sigma, U.S.A.) in 0.9% saline (20 mg/ml) emulsified with an equal volume of Freund's incomplete adjuvant as an antigen into the left front knee according to previous methods.14,15 Four days later 3 rabbits were i.v. injected with 37 MBq 99m-Tc(V) DMSA and the other 3 with 99m-Tc-HIG. Scintigrams were obtained at 1, 3, 6 and 24 h. The regions of interest (ROIs) over arthritic (A) and contralateral (C) normal knees were compared to obtain A/C ratios. 

RESULTS 
The biodistributions of 99m-Tc(V) DMSA, 99m-Tc(III) DMSA and 99m-Tc-HIG in mice with abscesses are shown in Fig. 1a-c. The maximum uptake was observed in the kidneys with all the RPs compared to other organs. The max. absolute uptakes as % injected dose/g tissue were 2.64 +- 0.87 (24 h), 36.1 +- 7.8 (6 h) and 24.5 +- 6.3 (6 h) for 99m-Tc(V) DMSA, 99m-Tc(III) DMSA and 99m-Tc-HIG, respectively. Most of the radioactivity administered was excreted by the kidneys in the case of 99m-Tc(V) DMSA compared to 99m-Tc(III) DMSA. The urinary excretion values not shown on the graphs were 116.1 +- 15.l, 120.4 +- 29.1 and 90.4%/g in the first hour for 99m-Tc(V) DMSA, 99m-Tc(III) DMSA and 99m-Tc-HIG, respectively. 
 The A/L, A/B, A/M, A/I and A/K ratios obtained in biodistribution studies are shown in Fig. 2a-c. Higher ratios were obtained with 99m-Tc(V) DMSA than with the other agents. The max. A/M ratio was 9.46 +- 3.20 at 24 h with 99m-Tc(V) DMSA compared to 5.98 +- 1.17 obtained with 99m-Tc-HIG. The 6 h ratio for 99m-Tc(V) DMSA (6.00 +- 2.52) was also higher than that for 99m-Tc-HIG (3.62 +- 0.86). The higher SD's of NM obtained with 99m-Tc(V) DMSA may be due to individual variations in blood clearance and renal excretion. The abscesses were better visualized on scintigrams with 99m-Tc(V) DMSA starting at 1 h (Fig. 3) than with 99m-Tc(III) DMSA. The organs that were visualized were the kidneys and the urinary bladder. 
 Scintigrams of rabbits demonstrated arthritic joints with both 99m-Tc(V) DMSA and 99m-Tc-HIG (Fig. 4). There was less blood background with 99m-Tc(V) DMSA than with 99m-Tc-HIG during 1-6 h post-injection. The bone uptake more evident in 99m-Tc(V) DMSA images of the rabbits than in mice scintigrams is in line with earlier reported biodistribution studies.1-3 For both RPs AIC ratios obtained by ROIs on scintigrams of rabbits are presented in Fig. 5. A/C ratios of 99m-Tc-HIG were a little superior after 3 h and kept increasing up to 24 h while 99m-Tc(V) DMSA showed a decline after 3 h, indicating a wash-out of radioactivity from the joints involved. 

DISCUSSION 

Our results demonstrated the accumulation of 99m-Tc(V) DMSA in experimental abscesses and arthritis. Rapid blood clearance and excretion predominantly via the kidneys may be responsible for the higher abscess/other tissue concentration ratios than with 99m-Tc(III) DMSA and 99m-Tc-HIG. Due to the absence of abdominal (liver and intestines) radioactivity it has the potential of demonstrating lesions in this region compared to 99m-Tc-HIG. The only disadvantage may be the low absolute uptakes by the abscesses. The max, uptakes were 0.645 +- 0.268 (1 h), l.17 +- 0.03 (3 h) and 3.20 +- 0.94 (6 h) % injected dose/g for 99m-Tc(V) DMSA, 99m-Tc(III) DMSA and 99m-Tc-HIG, respectively. This can be attributed to faster renal excretion as was also reported with other small molecular weight complexes of 99m-Tc that are mainly excreted via kidneys.10,15,16 The abscess-to-other tissue concentration ratios obtained with 99m-Tc(V) DMSA are higher than those obtained with agents previously tested in our laboratory,10,15,16 but the A/C values in rabbits are somewhat lower.15,16 In two patients with active tuberculosis the quality of 99m-Tc(V) DMSA scintigrams was superior to those of 99m-Tc-citrate,ll More comparative studies are necessary to prove the superiority of this new radio-pharmaceutical. It is evident that 99m-Tc(III) DMSA is not suitable for this purpose because of its lower concentration ratios obtained compared to the other two agents. Furthermore, high renal cortical localization of 99m-Tc(III) DMSA results in an unacceptably high radiation dose to the kidneys, since an adult dose of 555-740 MBq is necessary in human application. 
The main localization mechamsm or all the three agents may be infiltration into the interstitial space due to increased capillary permeability. The increase in A/M ratios as time progressed may be a result of high protein binding of DMSA complexes reported earlier.2 They also therefore behave like labeled proteins such as 99m-Tc-HIG and are entrapped in the interstitial space due to their assumed large molecular weight. It is also possible that 99m-Tc DMSA complexes or 99m-Tc itself are bound to proteins at the site of inflammation. The higher concentration ratios obtained with the pentavalent compared to the trivalent state of Tc in DMSA complexes also suggest that a structural similarity to the phosphate ion might play a role in its localization. More studies are needed to elucidate the localization mechanisms. 
 Commercially available nonspecific radiopharmaceuticals that are currently used for imaging inflammatory lesions are either macromolecules themselves such as 99m-Tc-HIG or assume a large molecular weight after i.v. administration such as 67-Ga-citrate from which 67-Ga dissociates and binds to transferrin in plasma.17 They have prolonged blood clearance and high abdominal localization, necessitating delayed imaging. Small molecular weight complexes such as 99m-Tc-citrate, 15 99m-Tc-DTPA, 10,18 99m-Tc-glucoheptonate,19 and 99m-Tc(V) DMSA may be better alternatives especially for lesions located in the abdominal region. They need to be further evaluated in comparative studies. 
 In conclusion, 99m-Tc(V) DMSA is preferred to 99m-Tc-HIG, because of the higher concentration ratios attained earlier, lower blood background, absence of radioactivity in abdominal organs such as the liver and intestines and lower cost, but it should be compared to other renal agents in clinical application to find the best radiopharmaceutical for imaging inflammatory lesions in man. 

ACKNOWLEDGMENT 

This work was supported by the Turkish Scientific and Technical Research Council (TUBITAK) (Project No: SBAGD-AYD-47). 

REFERENCES 

l. Yokoyama A, Hata N, Horiuchi K, et al. The design of a pentavalent 99m-Tc-dimercaptosuccinate complex as a tumor imaging agent. Int J Nucl Med Biol 12: 273-279, 1985. 
2. Chauhan UPS, Babbar A, Kashyap R, et al. Evaluation of a DMSA kit for instant preparation of 99m-Tc(V)-DMSA for tumour and metastasis scintigraphy. Nucl Med Biol 19: 825-830, 1992. 
3. Watkinson JC, Allen SJ, Laws DE, et al. The pharmacokinetics and biodistribution of technetium-99m(V) dimercap-tosuccinic acid in an animal model. J Nucl Med 32: 1235-1238, 1991. 
4. Ohta H, Endo K. Fujita T, et al. Imaging of soft tissue tumors with Tc(V)-99m dimercaptosuccinic acid. A new tumor-seeking agent. Clin Nucl Med 9: 568-573, 1984. 
5. Watkinson JC, Larazus CR, Mistry R, et al. Technetium-99m(V) dimercaptosuccinic acid uptake in patients with squamous carcinoma of the head and neck-Experience in imaging. J Nucl Med 30: 174-180, 1989. 
6. Kashyap R, Babber A, Sahai I, et al. Tc-99m(V) DMSA imaging. A new approach to studying metastases from breast carcinoma. Clin Nucl Med 17: 119-122, 1992. 
7. Kobayashi H. Sakahara H, Hosono M, et al. Soft tissue tumors: Diagnosis with Tc-99m(V) dimercaptosuccinic acid scintigraphy. Radiology 190: 277-280, 1994. 
8. Blower PJ, Sing J, Clarke SEM. The chemical identity of pentavalent technetium-99m-dimercaptosuccinic acid. J Nucl Med 32: 845-849, 1991 . 
9. Ohta H, Ishii M, Yoshizumi M, et al. A comparison of the tumor-seeking agent Tc-99m(V) dimercaptosuccinic acid and the renal imaging agent Tc-99m dimercaptosuccinic acid in humans. Clin Nucl Med 10: 167-170, 1985. 
10. Ercan MT, Unlenen E. Accumulation of some small molecular weight complexes of 99m-Tc in experimental abscesses. Nucl Med Biol 21 : 143-149, 1994. 
11 . Gulaldi NCM. Bayhan H, Ercan MT, et al. The visualization of pulmonary tuberculosis with Tc-99m(V) DMSA and Tc-99m citrate in comparison to Ga-67 citrate. Clin Nucl Med 20: 1012-l014, 1995. 
l2. Sampson C. Preparation of Tc-99m(V) DMSA. Nucl Med Commun 8: 184-185, 1987. 
13. Babber A, Kashyap R, Chauhan UPS. A convenient method for the preparation of 99m-Tc-labelled pentavalent DMSA and its evaluation as a tumor imaging agent. J Nucl Biol Med 35: 100-104, 1991. 
14. Consden R, Doble A, Glynn LE, et al. Production of a chronic arthritis with ovalbumin. Its retention in the rabbit knee joint. Ann Rheum Dis 30: 307-315, 1971 . 
15. Ercan MT, Aras T, Unlenen ES et al. 99m-Tc-Citrate versus 67-Ga-citrate for the scintigraphic visualization of inflammatory lesions. Nucl Med Biol 20: 881-887, 1993. 
16. Ercan MT, Unlenen E, Aktac A. 99Tc-m-glutathione for imaging inflammatory lesions. Nucl Med Commun 15: 533-539, 1994. 
l7. Tzen KY, Oster ZH, Wagner HN, et al. Role of ironbinding proteins and enhanced vascular perrneability in the accumulation of gallium-67. J Nucl Med 21: 31-35, 1980. 
l8. Hosain F, Haddon MJ. Hosain H, et al. Radiopharmaceuticals for diagnosis and treatment of arthritis. Nucl Med Biol 17: 151-155, 1990. 
19. Roizenblatt J, Buchpiguel CA, Menguetti JC, et al. Quantification of ocular inflammation with technetium-99m glucoheptonate. Eur J Nucl Med 18: 955-958, 1991 .