99mTc(Cu)—mannitol complex: a new agent for dynamic renal function studies

Nucl. Med. Eiol. Vol. 16, No. 3, pp. 283-289, ht. J. Radial. Appl. Instrum. Part B Printed in Great Britain. All rights reserved

0883-2897/89

1989

53.00 + 0.00

Copyright 0 1989Pergamon Press plc

99mTc(Cu)-Mannitol Complex: a New Agent for Dynamic Renal Function Studies U. P. S. CHAUHAN,* J. CHANDER, P. MISHRA, R. C. SARIN, R. V. NARAYANAN and B. SINGH Institute of Nuclear Medicine and Allied Sciences, Probyn Road, Delhi-l 10007, India (Received 3 June 1988) Mannitol has been labelled with WmTcby using cuprous chloride as a reducing agent. Blood and kidney clearance of 99mTc(Cu)-mannitol was slightly faster than that of %Tc(Sr+DTPA in rat and maximum radioactivity ratio of kidneys to blood was 84.6 at 5 min. A comparative study of 99”Tc(Cukmannitol, 99”Tc(Sn)-DTPA was made in rabbits by taking serial images of kidneys and bladder with a y camera. Results show superiority of 99mTc(Cu)-mannitol over other agents for dynamic renal function studies.

Introduction Among dynamic acquired

99”Tc-labelled radiopharmaceuticals for renal function studies 99”Tc(SnkDTPA has a wide recognition (Chervu and Blaufax,

1982; Hosain, 1974; Chervu er al., 1977). However the quality of the complex is of utmost importance because even a minor deterioration that may not affect scans would reduce renal clearance (Hosain, 1974). Efforts to develop 99”Tc-radiopharmaceuticals to replace radioiodinated hippurate for renal tubular function studies were not successful until recently when CO, DADS (Klingensmith et al., 1985) and triamide mercaptide ligands were developed (Kasina et al., 1986; Fritzberg et al., 1986). Among them mercapto acetyl glycyl glycyl alanine has been reported most promising (Eshima et al., 1987). On the other hand, “mTc-p-[(bis carboxy methyl)-amino methyl carboxy aminolhippuric acid (PAHIDA) has been found to be close to 99mTc-DTPA in its kinetics, though the agent structurely resembles hippurate (Summerville et al., 1987). A major modification in the labelling of DTPA with 99mT~ was made by substituting cuprous chloride for stannous chloride as a reducing agent (Chervu et al., 1977). The new complex *Tc(Cu)-DTPA claimed to meet more criteria stipulated for a GFR agent and suggested the use of cuprous chloride as a reducing agent in the preparation of 99”Tc-labelled radiopharmaceuticals. Preparation of 99mTc(Sn)-mannitol and its potential application for kidney imaging has been reported by us earlier (Subramanian et al., 1977). However, renal clearance of the agent was not at a sufficiently fast rate to use it for dynamic renal function and GFR studies. When cuprous chloride was substituted for *Author for correspondence.

stannous chloride in the above reaction, the new complex showed fast renal clearance and its suitability for use in dynamic renal function studies. This paper reports the method of preparation of 99mTc(Cu)-mannitol, and its biodistribution and renal clearance characteristics.

Materials and Methods All the chemicals used were of A.R. grade unless specified. pmTc]pertechnetate was extracted from 99Mo (Supplied by BARC, Bombay) by using freshly distilled methyl ethyl ketone (MEK). Radionuclidic, radiochemical and chemical purities were ensured before its use. Preparation of 9h Tc ( CU)-rnunnitol

Approximately 100 mg mannitol and 4 mg of cuprous chloride were dissolved in 2 mL of water by heating at 80°C for 2 min and shaking. The solution was diluted to 20 mL with water. An aliquot of 2 mL of this diluted solution was taken and its pH was adjusted to between 6 and 6.5 by adding a requisite volume of 10% sodium bicarbonate solution. Labelling of mannitol with 99mTcwas achieved by mixing the above preparation with 3-5 mL of 99mT~04 solution in physiological saline. The preparation was sterilized by filtering through 0.22~ millipore membrane filter (Millipore Corporation, U.S.A.), although the preparation was stable to autoclaving. Preparation of mTc(Sn)-mannitoi

The radiopharmaceutical was prepared by the method described for the preparation of 99mTc(Cuk mannitol with the only change that an equivalent 283

U. P. S.

284

amount of stannous cuprous chloride.

chloride

was substituted

CHALJHANet

for

Preparation of pATc(Sn)-DTPA

Approximately 80 mg of DTPA and 8 mg of stannous chloride were made to a clear solution in 2 mL of water by heating at 80°C for 2 min with shaking. The solution was cooled to room temperature and diluted 10 times with water. An aliquot of 2mL of diluted solution was taken separately and its pH was adjusted to 66.5 by adding an appropriate volume of 1N NaOH. 9h”Tc(Sn)-DTPA was prepared by mixing 24 mL of 99mTc0; solution in physiological saline as described for 99”Tc(Cukmannitol. Radiochemical purity and stability tests

Radiochemical purity and stability tests of the radiopharmaceuticals were carried out by ascending paper (Whatman 3 MM) chromatography using 100% acetone and 85% methanol as solvents. Radiochemical purity of the radiopharmaceutical preparation was expressed as percentage of total radioactivity applied on the chromatogram. Stability of the various agents was examined by measuring radiochemical purity of the agents after storing them for different time intervals at room temperature. Characterization of DTPA complexes

*Tc-labelled

mannitol

and

Horizontal paper (Whatman 3 MM) electrophoresis was carried out in 50 mM vemol buffer pH 7.0 at a potential gradient of 15V/cm for 90min. After drying the paper, it was scanned under a y camera to identify each of the radiopharmaceuticals. Blood protein binding

To 1 mL of heparinized human blood 0.1 mL of 99”Tc(Cu)-mannitol was mixed and the contents were incubated at 37°C for a period of 15 min. Plasma and red blood cells were separated by centrifugation. To 0.1 mL plasma, 1 mL physiological saline and 1 mL 10% cold trichloroacetic acid (TCA) were added. After mixing the contents, the protein precipitate was separated by centrifugation. Radioactivity was measured in both the precipitate and supernatant. The plasma protein bound activity was expressed as the fraction of preparation in per cent of the total activity present in blood.

al.

Lipophilicity

An aliquot of 0.1 mL of 99mTc(Cu)-mannitol preparation was added to a mixture of 2 mL of ethylene dichloride and 2 mL of physiological saline, The contents were mixed thoroughly and allowed to stand for 15 min to separate the two layers. Aliquots were taken from both the layers for measuring radioactivity. Lipophilicity of the radiopharmaceutical was expressed as the ratio of the radioactivity in ethylene dichloride fraction to that in aqueous phase. Biological distribution and organ specificity

A group of 25 healthy male rats (Sprague-Dawley) weighing 120-125 g were randomly divided into five groups: each group of five rats. An equal dose (1 PCi) of 99mTc(Cu)-mannitol preparation in 0.1 mL was intravenously administered through the tail vein to each rat. At 2, 5, 15, 30 and 60 min after injecting the radiopharmaceutical, the animals were guillotined (one group of rats per time period). Blood was collected separately. Liver, kidneys and heart were removed, made free from adhering tissues and blood before measuring the radioactivity and weighing. The actual amount of radioactivity administered to each animal was calculated by subtracting the activity left in the tail from the activity injected. Radioactivity accumulated in each organ was expressed as per cent administered dose. Total volume of blood was calculated as 7% of the body weight. For the purpose of organ distribution and kidney clearance of radioactivity, data per gram tissue as the per cent of administered dose were calculated. Dynamic renal function study

Dynamic renal function study was carried out in healthy rabbits after administering the radiopharmaceutical (100 FCi) through the ear vein. A series of scans of neck to bladder region were taken between 0 and 60 min by using a y camera (ZLC-37 Siemens LFOV) to examine the kidney specificity and renal clearance. Radioactivity seen in the kidneys and the bladder at various time intervals was also measured to evaluate the renal excretion kinetic of the radiopharmaceuticals.

Results Data on radiochemical purity and stability of 99mTc(Cu)-mannitol, 99mTc(Snkmannitol and 99mTc-

Table 1. Radiochemical purity and stability of WmTc(Cu)-mannitol, *Tc(Sn)-mannitol and %Tc(SnbDTPA. The preparations were subjected to ascending paper chromatography by using (A) 100% acetone and (B) 85% methanol as mobile phases. In both the solvent systems the pharmaceuticals remained at ihc point of application. Data are expressed as per cent radioactivity applied *Tc(SnFmannitol Duration IOmin lh 2h 3h

4h 5h

A

B

A

B

A

B

98.10

97.90

98.29

99.10

99.02

98.76

97.91 99.97 97.85 97.27 95.92

91.83 97.81 97.20 96.85 96.72

97.97 91.91 97.78 97.57 97.57

98.52 99.16 98.04 97.81 97.96

98.88 98.83 98.71 91.57 -

98.12 97.81 97.17 96.45 -

285

99mTc-labelled mannitol

(St+DTPA

as obtained

graphy are presented

by ascending

chromato-

in Table 1. Only two radioactive

spots were detected on the chromatograms by using either of the solvent systems with all the three radiopharmaceuticals. The radiopharmaceuticals were identified with the RF O-0.1, whereas %TcO; was identified with RI 0.9-1.0. Radiochemical purity of the three radiopharmaceuticals within 10 min of labeling with the radioisotope was greater than 98% radiochemical purity even after 4 h storing at room temperature indicates that the radiopharmaceuticals are sufficiently stable for kidney imaging and dynamic renal function studies. Electrophoretic behaviour of 99”Tc(Cukmannitol, 99”Tc(Sn)-mannitol, 99mTc(CubDTPA, 99mTc(Sn)DTPA and %TcO; are showing in Fig. 1. The electrophorogram exhibits different mobility of the various agents under similar conditions which suggests that the Tc-complexes are different when SnCI, was replaced by CuCl. clearance of Organ specificity and renal 99mTc(Cu)-mannitol in rats with time is shown in Table 2. Location of 46.8% radioactivity in the kidneys after 2 min of its administration clearly demonstrates its preferential kidney uptake. Most of the remaining radioactivity was in the blood, whereas heart and liver picked up only a small percentage of radioactivity. At 5 min two thirds the amount of radioactivity was located in kidneys. Blood, heart and liver together had only 18% of the injected dose. The remaining radioactivity was most likely excreted through the kidneys by this time. Fast renal clearance of the agent would be evident from organ distribution data at 15, 30 and 60 min. At 15 min only 45% of the administered dose of radioactivity was found in the various organs out of which two thirds was located in the kidneys. Although 80% of the injected radioactivity was cleared at 6Omin, some radioactivity was still present in blood and kidneys. Pattern of kidney excretion of 99”Tc(Cu)-mannitol and ratio of radioactivity in kidneys to blood at various time intervals after administering the agent are shown in Fig. 2. The maximum amount of radioactivity in kidneys was recorded at 5 min but was reduced to half at 9 min. Exponential decrease of the radioactivity in kidneys with time was noted thereafter. Kidneys to blood radioactivity ratio with time also revealed a more or less similar pattern. Data on distribution of radioactivity in the various blood components in vitro demonstrated that the major amount of radioactivity was unbound.

Whereas 8% radioactivity was bound to plasma proteins, about 1% was attached to RBC. These data explain why 6% radioactivity was seen in blood even at 60min after injecting the agent to the animal. Lipophilicity of 99”Tc(Cu)-mannitol was studied by partitioning the preparation between methylene dichloride and physiological saline. Very low lipophilicity of the agent (0.006) may be a contributing factor to its preferential renal excretion. of Kidney specificity and renal clearance y9mTc(Cu)-mannitol in rabbit is shown in Fig. 3. Kidney uptake and its excretion through them at a fast rate would be evident from the serial of scans of neck to bladder region. The maximum amount of radioactivity (t,,,) in the kidneys was seen at 2 min 10 s which was reduced to its half value (tliz) at 11 min 10 s (Fig. 4). When under similar conditions the performance of 99mTc(Sn)-mannitol was evaluated (Figs 5 and 6), the clearance of the agent was much slower, although the maximum amount of radioactivity was accumulated at about 3 min. However, the accumulated radioactivity was not reduced to half even at 80 min after drug administration. 99mTc(Sn)-DTPA was also evaluated under the conditions similar to those used for gR”Tc(Sn)-mannitol (Figs 7 and 8). The biological behaviour of this agent was very much similar to that of 99mTc(Cukmannitol. However, time of maximum accumulation in kidneys (t,,,) (3 min) and (t,,,) 50% reduction of the activity thereafter (14 min) were greater than those observed for 99mTc(Cu)-mannitol.

Discussion A noticeable difference in the electrophoretic mobility and renal excretion of gR”Tc(Cu)-mannitol and 99mTc(Sn~mannitol suggests the significance of selecting an appropriate reducing agent since it can alter or modify the biological behaviour of the radiopharmaceutical probably due to the change in chemical nature of the complex(es) formed. A similar alteration in the electrophoretic mobility and biological behaviour was observed when Cu+ was substituted for Sn*+ in the preparation of 99mTc-GHA. The new agent was found to concentrate in the spleen rather than in the kidneys (Chauhan et al., 1987). The mechanism of such alterations in the biological behaviour of the radiopharmaceutical due to a reducing agent is not known. It could be due to either different reducing potencies of the reducing agents or their participation in the complex formation. In fact cop-

Table 2. Organ distribution of PR”Tc(Cu)-mannitol with time in rat. Each value is the mean f SE of five experiments. Values arc cxpnssed as per cent radioactivity of the administered dose in the whole organ Time after administration Tissue Blood Heart Liver Kidneys

2 min 36.89 * 3.69 4.13 *0.37 12.00 f 1.09 46.84k3.10

5 min 10.95 * 0.61 f 7.29 f 66.14 f

1.48 0.08 0.75 2.43

15min 8.93 f 0.70 f 5.35 f 31.18 *

1.19 0.10 0.42 2.26

30 min

60 min

6.38 f 0.33 0.56 k 0.0s 1.91 *0.51 14.48 f 1.50

6.68 * 0.64 0.74 f 0.11 1.50*0.15 10.89 * 0.33

u. P. s. CHAuHANet

al.

40 r 36

-

32

-

26

-

T

24-

5! x

20-

u

16

0

Bladder

10

20

30

40

50

60

70

00

t lmin)

Fig. 6. Renal excretion of 99mTc(Sn)-mannitolin a normal rabbit after intravenously administering IOOpCi of the agent. lime

lmin)

Fig. 2. (a) Kidney clearance of the radiopharmaceuticals with time. (b) Kidney to blood radioactivity ratio with time. Normal rats were intravenously injected with 1PCi of gA”Tc(Cukmannitol and radioactivity Per gram tissue at different time intervals was calculated as the per cent injected dose. Each value is the mean f SE of five rats. per has been suggested as the part of *“Tc-DTPA when cuprous chloride was used as a reducing agent in the preparation of the above complex (Chervu et 24r

0

4

6

12

16

20

t

24

26

32

34

(mln)

Fig. 4. Renal excretion of 99mTc(Cu)-mannitolin a normal rabbit after intravenously administering 1OOfiCi of the agent.

al., 1977). However, conclusive evidence of whether a reducing agent is part of the complex has yet to he produced. The metabolic pathway of copper administered orally or intravenously has heen reported (Chervu and Stemlieu, 1974). An amount upto 3 mg of cuprous chloride did not cause any renal damage prepared by the method in rat. *“Tc(Cu~mannitol described here contains only about lSOpg/mL concentration of cuprous chloride and therefore is unlikely to cause any untoward reactions in patients. The renal excretion of the WmTc(Cubmannitol is not inhibited in probencid treated rats (data not shown). It suggests that 99”Tc(Cu)-mannitol is cleared by glomerular filtration as in the case of DTPA, the most widely used 99mTc-labelled radiopharmaceutical for dynamic renal function studies (Chervu and Blaufox, 1982). The plasma clearance and urinary execretion of WmTc(SnkDTPA have been studied thoroughly (Hauser er al., 1970; Atkins et al., 1971; Klopper et al., 1972; McAfee et al., 1979). Values of 1,, and tr,r of g9mTc(Cu~mannitol for kidneys are slightly better compared to those for 99mTc(SnkDTPA. Exclusive renal excretion of the radiopharmaceutical may be appreciated due to its very poor lipophilicity. Human plasma protein binding of 99mTc(Cu)-mannitol is greater as compared to l&5.9% (average 3.7%) reported for WmTc(Sn)DTPA (Klopper et al., 1972). However, only 6% radioactivity in blood at 60 min after the administration of 99mTc(Cukmannitol compares well with 8% retention of radioactivity in blood when 99mTc(Sn)DTPA was used in rat (McAfee et al., 1979) and rabbit (Arnold et al., 1975). It is of interest to point out that the maximum ratio of radioactivities in kidneys to blood (per g of tissue)

.

.’

1

2

3

4

5 10 min

5 min

Immediate

(A)

(B)

I

Ic

‘1,

50

35 min

20 min

Fig. 1. Horizontal paper electrophoresis of 99”Tc-labelled radiopharmaceuticals. (A) 1, wmTc(Cu)mannitol; 2, %Tc (Sn)mannitol; 3, wmTc(Cu)DTPA; 4, 99”Tc(SnkDTPA; 5, %TcO,. (B) I, *Tc(Sn)EHDP; 2, 99”Tc(Cu~EHDP; 3, wmTc(Sn~HA, 4, 99”Tc(Cu)-GHA; 5, %TcO.,. (C) 1, wmTc(Sn)MDP; 2, 99mTc(Cu)-MDP, 3, %TcO,.

a

8

I

min

* Ir

I, 65

min

60

min

Fig. 5. Scans of neck to bladder region of a normal rabbit of different time intervals after intravenously administering 100 pCi of 9PmTc(Cutmannitol

a

a 1 min

2.5

min

4.5

a

min

2 min

u

v

a

#

a

*

0

Q

r)

0

c

a

20

min

45 gg m

mln

Tc- Cu-mannitol

I f

I,

25 min 10 min

35 min

a

4P

* 10 min

5 min

60

(rabbit

min

40

1

min

20

min

30 min

50

mitt

60

ggmTc-Sn-DTPA

(rabbit

min

1

Fig. 7. Scans of the neck to bladder region of a normal rabbit at different time intervals after intravenously administering 100 nCi of gR”Tc(Sn)-DTPA.

Fig. 3. Scans of neck region of a normal rabbit at different time interval after intravenously administering 1OOnCi of 9R”Tc(Cukmannitol. 287

289

gR”Tc-labeIled mannitol

this study. Our thanks are also due to Mr Vinod Behari and Mr Krishan Sawroop for the technical assistance.

References Arnold, R. W.; Subramanian, G.; McAfee, J. G.; Blair, R. J.; Thomas F. F. Comparison of %Tc complexes for renal imaging. J. Nucl. Med. 16: 357; 1975. Atkins, H. L.; Cardinale, K. G.; Eckelman, W. C.; Hauser, W.; Klopper, J. F.; Richards, P. Evaluation of %Tc-DTPA prepared by three different methods. Radiology 98:

674; 1971.

Chauhan, U. P. S.: Dhall, J. C.; Pushpa, Mishra; Babbar, A. K.; Kaushik, S. K.; Chopra, M. K. 9R”Tc-Cu-GHA: A new radiopharmaceutical for spleen scintigraphy. Ind. J. Nucl. Med. 2: 22; 1987. t lmin) ssmTc-Sn-DTPA

Fig. 8. Renal excretion of WmTc(Sn)-DTPA in a normal rabbit after intravenously administering 100 PCi of the agent.

was 84.6 at 5 min after WmTc(Cu)-mannitol administration in rat. Such a figure for 99”Tc(Sn)-DTPA is not available in the rat. However such ratios for 99”Tc(Sn)-DTPA were only 5 at 3 min in dog (Chervu et al., 1977) and 4.3 at 60 min in rabbit (Arnold et al., 1975), which are much less than the ratios of 13.8 and 13.2 observed at 2 and 60 min respectively in mice in this study (Fig. 2). Such data not only demonstrate specificity of 99”Tc(Cu)-mannitol for kidneys and suitability for dynamic renal function studies but also its superiority over other WmTc-labelled agents used for this purpose. It may be appropriate to suggest that the use of a reducing agent in the preparation of 99mTc-mannitol has a crucial role in the complex formation in addition to the reduction of Tc from 7 to a lower valency state. NMR spectroscopy and X-ray crystallography may shed some light on the chemical difference between the Tc-mannitol complexes formed when cuprous chloride and stannous chloride are used respectively. However, the above techniques would require larger amounts of the preparations which could be achieved only by using 9%c instead of *Tc.

Chervu, L. R.; Blaufox, M. D. Renal radiopharmaceuticals: An update. Semin. Nucl. Med. 12: 224; 1982. Chervu L. R.; Lea, H. B.; Goyal, G.; Blaufox, M. M. Use of 99”Tc-Cu-DTPA complex as a renal function agent. J. NW{. Med. 15: 1011; 1977. Chervu, L. R.; Sternlieb, I. Dosimetry of copper radionuclides. J. Nucl. Med. 15: 1011; 1974. Eshima, D.; Taylor, Jr A.; Fritzberg, A. R.; Kasina, S.; Hansen, L.; Sorenson, J. F. Animal evaluation of %Tc triamide mercaptide complexes as potential renal imaginirm agents. J., Nucl. Med. 28: 1180, 1987. Fritzberg, A. R.: Kasina, S.; Eshima, D.; Johnson, D. L. Evaluation of a new *Tc renal imaging agent. %Tc mercopto acetyl glycyl glycine (MAG 3) as a hippuran replacement. J. NucI. Med. 27: 111; 1986. Hauser, W.; Atkins, H. L.; Nelson, K. G.; Richards, P. P9”Tc-DTPA: A new radiopharmaceutical for brain and kidney imaging. Radiology 94: 679; 1970. Hosain, F. Quality control of sr’“‘Tc-DTPA by double tracer clearance technique. J. Nucl. Med. 15: 442; 1974. Kasina, S.; Fritzberg, A. R.; Johnson, D. L.: Eshima, D. Synthesis of diamide dimercaptide ligands and their %Tc complexes distribution relationships and renal radiopharmaceutical potential. J. Med. Chem. 29: 1933; 1986. Khngensmith, III W. C.; Tyler Jr, H. N.; Marsch, W. C.; Hanna. G. H.: Fritzbern. A. R.: Halt. S. Effect of hydration and dehydratioi’and on Technetium-99m CO, DADS, Renal studies in normal volunteers. J. Nucl Med. 26: 875; 1985. Klopper, J. P.; Hauser, W.; Atkins, H. L.; Eckelman, W. C.: Richards. P. Evaluation of %Tc-DTPA for the measurement of glomerular filtration rate. J. Nucl Med. 13: 107; 1972. McAfee, J. G.; George, G.; Atkins, H. L.; Kirchences. P. T.: Reba. R. C.: Blauforz. M. D.: Smith. E. M. Biulonical distribution and excretion of DTPA with %Tc and ii%i. J. Nucl. Med. 20:

1273; 1979.

Subramanian, G.; Chander, J.; Singh, M. V.; Singh, B. 99mTc-labelledradiophannaceuticals for renal scintigraphy. Ind. J. Radiol. 31: 22; 1977.

Acknowledgements-The authors are grateful to Maj Gen (Retd) N. Lakshmipathi, Consultant and Professor V. K. Jain, Director of the Institute for their kind interest in

Summerville, D. A.: Packard, A. B.; Bartynske, B.; Lim, K. S.; Chervu, L. R.; Treves, S. T. Evaluation of the renal clearance of %Tc-PAHIDA in dogs. J. Nucl. Med. 28: 907: 1987.