99mTc-labeling kinetics of four thiol-containing chelators and 2-hydrazinopyridine: Factors influencing their radiolabeling efficiency

Appl. Radiat. lsot. Vol.48, No. 8, pp. 1103-1111, 1997 ,~ 1997The Du Pont Merck PharmaceuticalCompany Publishedby ElsevierScienceLtd. Printedin Great Britain 0969-8043/97 $17.00+ 0.00

Pergamon

PII: S0969-8043(97)00033-X

99mTc-labeling Kinetics of Four Thiol-containing Chelators and 2-Hydrazinopyridine: Factors Influencing Their Radiolabeling Efficiency S. L I U * , D. S C O T T E D W A R D S , A. R. H A R R I S a n d P. R. S I N G H The DuPont Merck Pharmaceutical Company, Radiopharmaceuticals Division, 331 Treble Cove Road, North Billerica, MA 01862, U.S.A. (Received 18 October 1996; Accepted 2 March 1997)

The relative °gmTc-labelingefficiency of several potentially tetradentate thiol-containing chelators was studied by competing them with glucoheptonate in the [99mTc]glucoheptonatecomplex, and was compared to that of 2-hydrazinopyridine (HYPY), a model compound for HYNIC (hydrazinonicotinamide). The thiol-containing chelators in their unprotected forms include 4,5-bis(mercaptoacetamido)pentanoicacid (H4LI), N-[2-(mercapto)propionyl]glycylglycylglycine(H4L2), 2-(mercapto)ethylaminoacetyl-L-cysteine (H3L3), and N,N'-ethylenediyl-bis-L-cysteine diethyl ester (H3L4). There are several factors that influence the labeling efficiency of a chelator. These include donor atoms, chelator concentration, and reaction conditions such as temperature and reaction time, and pH of the reaction media. In all the cases, higher chelator concentration produces better radiolabeling yields. Heating at 100°C for 30 min is required for the successful ~mTc-labelingof N:S2 diamidedithol (H4LI) and NsS triamidethiol (H4L2), while the N:S2 monoamide monoaminedithiol (H3L3) can be well labeled under milder conditions. For the N252 diaminedithiol (H3L4), the ligand exchange could be completed within 60 min at room temperature. It is clear that substitution of an amine-N for an amide-N enhances the rate of 99mTc- labeling. The reaction of HYPY with ~mTc]glucoheptonatewas carried out by heating the reaction mixture at 50°C for 30 min under acidic conditions (pH 4-5). The radiolabeling efficiencyof HYPY using glucoheptonate as coligand is better than that of H4LI-H3L3, and is comparable to that of H3L4. Therefore, HYNIC and diaminedithiols are the candidates of choice as bifunctional coupling agents in labeling small biologically active moleculeswith very high potency. © 1997The Du Pont Merck Pharmaceutical Company Published by Elsevier Science Ltd

Introduction There is great current interest in the development of receptor-based target-specific radiopharmaceuticals by the 99~Tc-labeling of biologically active small molecules, such as chemotactic peptides for imaging focal infection (Babich et al., 1993; Fischman et al., 1994; Babich and Fischman, 1995), and somatostatin and its analogs for tumor imaging (Maina et al., 1994). We have been actively pursuing a research program towards developing a thrombus imaging agent by labeling cyclic glycoprotein lib/Ilia (GPllb/llIa) receptor antagonists with 99mTc(Barrett et al., 1994; Harris et al., 1994; Barrett et al., 1995; Barrett et al., 1996; Edwards et al., 1997; Liu et al., 1996a; Liu et al., 1996b). Since these small peptides are very potent, with IC50 values in the nanomolar range (Jackson et al., 1994), the 99"Tc-labeling must be accomplished with high specific activity. There*To whom all correspondance should be addressed.

fore, the choice of a bifunctional chelator is very important for the success of the radiolabeling. Various chelators have been used in labeling proteins and small biologically active molecules with 99mTC and IS6Re. These include DTPA (Lanteingne and Hnatowich, 1984), N3S triamidethiols (Schroff et al., 1990; Breitz et al., 1992; Liu and Edwards, 1995), N2S2 diamidedithiols (Fritzberg et al., 1988a; Eary et al., 1989; Kasina et al., 1991; Liu and Edwards, 1995), N2S2 monoamidemonoaminedithiols (Kung et al., 1995), N2S2diaminedithiols (Baidoo and Lever, 1990; DiZio et al., 1991; Eisenhut et al., 1991; DiZio et al., 1992; Baidoo et al., 1994; Eisenhut et al., 1996), BATOs (Linder et al., 1991a, 1991b), PnAO (Maina et al., 1994), tetra-amines (Maina et al., 1995), and hydrazinonicotinamide (HYNIC) (Abrams et al., 1990; Schwartz et al., 1991; Babich et al., 1993; Babich and Fischman, 1995; Fischman et al., 1994). However, there is very little comparative data on these chelators performed under similar controlled conditions. In order to choose a suitable

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bifunctional coupling agent (BFCA), we have examined the relative labeling efficiency of several potentially tetradentate thiol-containing chelators (Fig. 1) by competing them with glucoheptonate in the [~mTc]glucoheptonate complex. We also studied the labeling efficiency of 2-hydrazinopyridine (HYPY), which is a model compound for HYNIC, using glucoheptonate as coligand. The results of this study allow us to compare the labeling efficiency of various bifunctional coupling agents.

Experimental Materials

Chemicals were purchased from Aldrich Chemical Co. and were used as received. Na99mTcO4 was obtained from a commercial DuPont Merck (North Billerica, MA) 99Mo/99mTcgenerator. The Glucoscan" kit used is also a commercial product from DuPont Merck. Deionized water was obtained from a Millipore MilliQ Water System and was of > 18 Mf~ quality. The benzoyl-protected HaL1 and HAL2, acetamido-protected HAL2, and trityl-protected ethyl ester of H3L3 were prepared as previously reported (Liu et al., 1996b). H3L4 was prepared according to the literature method (Blondeau et al., 1967). 2-Hydrazinopyridine was used as its dihydrochloride salt. Methods

The radio-HPLC method 1 used a Hewlett Packard Model 1050 instrument and a Vydac C~ column (4.6 mm x 25 cm, 300 A pore size) at a flow rate of l mL min ~ with a gradient mobile phase from 100% A (10 mM phosphate buffer, pH 6), to 30% B (acetonitrile) at 15 min and 75% B at 25 min, and radiometric detection by a sodium iodide probe. The radio-HPLC method 2 used a Hewlett Packard

Model 1050 instrument, a Vydac C,8 column (4.6 mm × 25 cm, 300 ,& pore size) and a gradient mobile phase from 100% A (10 mM phosphate buffer, pH 6), to 50% B (acetonitrile) at 15 min and 75% B at 25 min. The instant thin layer chromatography (ITLC) method used Gelman Sciences silica-gel strips and a 1:1 mixture of acetone and saline as eluant. Preparation of chelator solutions

H~L4.2HC1 was dissolved in 0.025 M phosphate buffer (pH 7.5) to give a concentration of 2.5 × 10 ~ M. 2-Hydrazinopyridine dihydrochloride was dissolved in 0.025 M phosphate buffer (pH 5) and was diluted to 2.5 × 10 -3 M. The benzoyl-protected HaLl and HaL2 were dissolved in 1 N NaOH solution to give a concentration of 1.0 x 10 -2 M. The resulting solutions were heated at 80:C for 5 rain. To 1 mL of the solution above was added 1.5 mL of 0.025 M phosphate buffer (pH 7.5). The resulting mixture was neutralized to pH 7.5 using 1 N HCI, and then diluted to 2.5 × 10 -3 M using 0.025 M phosphate buffer. The H3L3 solution was prepared according to the method described previously (Liu et al., 1996b) with slight modification. 2-[(S-Tritylmercapto)ethylaminodacetyl]-S-trityl-L-cysteine ethyl ester (10 mg) was dissolved in TFA (2 mL) to give a bright yellow solution. The mixture was stirred at room temperature for 15 rain. Upon addition of triethylsilane (1 mL), the reaction mixture became clear, and was stirred at room temperature for another 10 min. TFA and excess triethylsilane were removed under vacuum to give a white solid residue. To the residue was added l mL of 1 N NaOH solution and 1.0 mL of 0.025 M phosphate buffer (pH 11). The mixture was heated at 80°C for 5 min. The pH was adjusted to ~ 9 using 1 N HCI. The

M~,~ i? 0 C OOH

/--~

~,~COOH

ooV H4L1

H4L2

EtOOC~ N / ~ N pCOOEt

HsL3

0

NH I

NH2 H3L4

HYPY

Fig. 1. Unprotected chelators used in this study.

Radiolabeling efficiency of chelators solution was filtered, and the filtrate was diluted to 2.5 x 10 -3 M using 0.025 M phosphate buffer. The freshly prepared solution was immediately used for further reactions. Preparation o f Glucoscan" kit

Generators with 24 + 2 h prior-elution times were used for the preparation of the [99mTc]glucoheptonate complex. To a Glucoscan" kit was added 1 mL of 99mTcO4- saline solution (containing ~ 200 mCi). The reaction mixture was allowed to stand at room temperature for 15-20 min, and then used for ligand exchange reaction within 60 min. General procedure .for the labeling efficiency studies

The amounts of the three solutions (chelator, Glucoscan", and buffer) used in these experiments are shown in Table 1. After reconstitution, the reaction mixture was heated at 100-C for 30 min (H4LI, H4L2, and H3L3), or heated at 50~C for 30 rain (HYPY) or allowed to stand at room temperature (for H3L4) for 60 min. The reaction solution was then analyzed by radio-HPLC and ITLC.

Results and Discussion In the last decade, a large number of radiolabeling techniques have been used to label proteins and peptides with 99mTc (Otsuka and Welch, 1987; Fritzberg et al., 1988b; Delmon-moingeon et al., 1991; Eckelman et al., 1989: Hnatowich, 1990a, 1990b; Srivastava and Mease, 1991). These techniques are usually divided into three main categories: direct labeling; the preformed chelate approach; and the indirect labeling approach. The direct labeling approach uses a reducing agent to convert a number of disulfide linkages in a protein into free thiols, which are able to bind the Tc very efficiently. The advantage of this approach is that it is easy to carry out. However, very little is known about the coordination chemistry of the Tc center. There is little control over the stability of the 99mTc complex or nonspecific binding. In addition, this method applies only to proteins or their fragments because many small molecules such as peptides do not have any disulfide bonds, or in some cases the disulfide bond is too critical for maintaining their biological properties to be reduced. The preformed chelate

Table I. Components levels in the reaction mixture Concentration (M) 2.5 x 10 4 1.25 × 10 4 2.5 x 10 -~ 1.25 x 10 -~ 2.5 x 10 ~

Chelator (/~L) 100 50 10 5 1

Glucooscan" (uL)

Buffer (,uL)

100 100 100 100 100

800 850 890 895 899

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approach involves formation of the ~gmTc complex with a bifunctional chelator, and conjugation of the [99mTc]chelator complex to a protein or peptide in a separate step on the tracer (99mTc) level. In this approach, the chemistry is better defined, and the peptide or protein is not exposed to the sometimes forcing conditions used to prepare the complex. However, the multi-step tracer (99mTc) level synthesis makes it very difficult to develop a kit formulation, thereby limiting its clinical application. In the indirect labeling approach, a bifunctional coupling agent is first attached to the peptide or protein to form a chelator-peptide (protein) conjugate. The radiolabeling can be achieved either by direct reduction of 99mTcO4 in the presence of a chelator-peptide (protein) conjugate or by ligand exchange with an intermediate 99mTc complex such a s [99mTc]glucoheptonate. This approach combines the ease of direct labeling with the well-defined chemistry of the preformed chelate approach. It is also easy to make a kit formulation using this approach. Therefore, the indirect labeling is the most practical approach for the development of peptide-based target-specific radiopharmaceuticals. Two methods are often used to label the chelatorpeptide (or protein) conjugate. In the one-step synthesis, the chelator-conjugated biologically active molecule is labeled by the direct reduction of [99mTc]pertechnetate with a reducing agent such as stannous chloride. The choice of reducing agent may affect the labeling kinetics by this method. In the twostep synthesis, an intermediate complex such as [~gmTc]glucoheptonate is formed and is then allowed to undergo ligand exchange with the chelator-peptide (or protein) conjugate. In both cases, the choice of a bifunctional coupling agent is critical for successful radiolabeling. An ideal chelator is that which forms stable technetium complexes with high radiochemical purity (RCP) and high specific activity under mild conditions by either the one-step synthesis or the two-step ligand exchange synthesis. In this study, we compared the 99mTc-labeling efficiency of five types of bifunctional coupling agents: NzS2 diamidedithiol (H4L1); N3S triamidethiol (H4L2): N2S2 monoamidemonoaminedithiol (H3L3); N_~S~ diaminedithiol (H3L4); and hydrazinonicotiamide (HYNIC). H4LI and H~L2 have been used for the 99mTc-labeling of proteins (Fritzberg et al., 1988a; Eary et al., 1989; Kasina et al., 1991) and small peptides (Liu and Edwards, 1995: Liu et al., 1996b) by the preformed chelate approach. HYNIC have been used to label proteins (Abrams et al., 1990: Schwartz et al., 1991) and small peptides (Babich et al., 1993; Fischman et al., 1994; Babich and Fischman, 1995) by the indirect labeling approach. We chose to use the ligand exchange method to study the relative labeling efficiency of H,LI H,L4 by competing them with the glucoheptonate in the [99mTc]glucoheptonate complex, which is often used

S. L i u et al.

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an intermediate complex to label proteins and their fragments. To compare the labeling efficiency of four thiol-containing chelators with that of HYNIC, we used HYPY as a HYNIC model compound and glucoheptonate as the coligand. The S-benzoyl group is usually deprotected by a base-catalyzed hydrolysis. It was reported that the benzoyl-protected MAG3, which is a close analog of the benzoyl-protected H4L2, could be completely hydrolyzed by heating in a pH 9 phosphate buffer at 100°C for 10 min (Bormans et al., 1995). We deprotected the S-benzoyl group by dissolving the benzoyl-protected chelator in 1.0 N NaOH and heating the resulting solution at 80°C for 5 min. Therefore, the benzoyl group is expected to be completely deprotected. The S-trityl group was deprotected by a cationic cleavage of the S-C bond in anhydrous trifluoroacetic acid with triethylsilane as the cation scavenger. Hydrolysis of the ester group in 1.0 N NaOH produces free H3L3. The unprotected chelators were immediately used for the radiolabeling studies. In order to select a suitable bifunctional coupling agent, and to find optimum radiolabeling conditions, we studied several factors that might influence the rate of the ligand exchange reaction. These include the labeling conditions (such as the pH value of the reaction media, and the reaction time and temperature), chelator concentration, and the set of donor atoms in the Tc-bonding group. The pH Value

The pH effect (Table 2) on the ligand exchange reaction was studied using a fixed chelator concentration (2.5 x 10 4 M), at which all four thiol-containing chelators can undergo complete ligand exchange with the glucoheptonate. It was found that the pH effect depends on the nature of donor atoms in a chelator. For H4L1 and H4L2, alkaline conditions (pH _> 7.5) are required for the successful radiolabeling (RCP _> 90%). H3L4 is well labeled at a pH range of 5.0-9.0. (At higher pH, the two ester groups will be hydrolyzed; thereby lowering RCP.)

Reaction time and temperature

In general, prolonged heating ( > 30 min) at 100°C is required for the N2S2 diamidedithiol (H4L1) and N3S triamidethiol (H4L2), particularly at lower concentration. Shorter reaction time and lower temperature may dramatically decrease the labeling yield. For the N2S2 monoamide monoaminedithiol (H3L3), the reaction temperature could be 70-100°C; but heating for 30 min is necessary for the complete ligand exchange. For the N2S., diaminedithiol (H3L4), the complex [99"TcO(L4)] is not stable at 100°C, particularly at pH > 7.0. Therefore, we cannot use the reaction conditions used for H4LI-H3L3. It should be noted that the reaction time for H4L4 to undergo complete ligand exchange depends on the chelator concentration. At concentrations higher than 1.25 x 10 -4 M the differences in rasdiolabeling efficiency of H4L4 were minimum between 30 and 60 min reactions. However, at lower concentrations (2.5 x 10 -5 M-2.5 x 10 -6 M) the longer reaction time produces better radiolabeling yield for the complex [99"TcO(L4)]. Chelator concentration

Since it is a ligand exchange reaction, the concentrations of both glucoheptonate and the chelator are important. If a whole Glucoscan R kit (containing 200 mg of sodium glucoheptonate) is used, it requires a high chelator concentration to complete the ligand exchange. If the concentration of glucoheptonate is too low ( < 20 mg mL-~), the Tc(V)--O core is not well stabilized and [99mTc]colloid will form. In this study, the concentration of glucoheptonate is fixed at 20 mg mL-~, which is only 1/10 of a Glucoscan" kit. In all the cases (H4L1-H3L4), the higher chelator concentration results in better radiolabeling (Table 3). The lower concentration limit for H4L1 is 1.25 x 1 0 - 4 M ( ~ 50 #g m L - ~). For H4L2, the lower concentration limit is 2.5 x 1 0 - 4 M (100~tgmL -~) while that for H3L4 is 1.25x 1 0 - S M ( ~ 5 / t g m L -~, Fig. 2). [99mTc]colloid formation was found to be minimal.

Table 2. The effects o f pH value on RCPs Compound H4LI

H4L2

H~L3

H3L4

Concentration (M) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

x x x × x x x x x x x x x x

l0 -4 10 -4 10 -4 10 -4 10 -4 10 -4 10 -a 10 -4 10 4 10 4 10 -4 10 -4 10 4 10 -4

Temperature ( C ) 100 100 100 100 100 100 100 100 100 100 RT RT RT RT

~Hydrolysis o f the ester groups produced lower RCPs.

pH l 1.0 9.0 7.5 5.0 I 1.0 9.0 7.5 9.0 7.5 5.0 11.0 9.0 7.5 5.0

Time (rain) 30 30 30 30 30 30 30 30 30 30 60 60 60 60

R C P (%) 96 -I-_4 95 _+ 2 90 + 3 80_-/-8 80 _+ 5 60 + 2 48 --I- 5 92 (n = I) 90 __. 3 65 ( n = I) 76 __ 3' 97 __. 3 99+2 98 + 2

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Table 3. Effects of chelator concentration on RCPs Compound

Concentration (M)

H4L1

2.5 1.25 .5 1.25 2.5 1.25 2.5 2.5 2.5 1.25 2.5 1.25 2.5 1.25 2.5 1.25 2.5 2.5 1.25 2.5 1.25

H4L2

H~L3

H~L4

x x x x × x x × × x × x × x ×

x × × × × ×

Temperature ( C )

10 4 10 4 10 -5 10 - : 10 6 10 -~' 10-4 10 -5 10 4 10 -4 10 ~ 10 -~ 10 4 10 -.4 10 -~ l0 ~ 10 -6 10 4 10 4 10 -~ 10 -~

pH

100 100 100 100 100 100 100

7.5 7.5 7.5 7.5 7.5 11.0 II.0

100

II.0

100 100 100 100 RT RT RT RT RT RT RT RT RT

7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5

Time (rain)

RCP (%)

30 30 30 30 30 30 30 30 30 30 30 30 60 60 60 60 60 30 30 30 30

91 + 3 85 + 3 72 _+ 5 60 + 5 30_+8 93 ___2 75 + 5 25+4 90 + 3 90 + 4 85_3 70_+4 95 + 2 95 -+ 2 90 -+ 3 85 -+ 5 60 -+ 5 95___2 95 _+ 2 70 _+ 3 64 _+ 5

~The reaction mixture contains 0.5 mL of H4L2 solution in phosphate buffer (pH = I I), 0.1 mL of Glucoscan" solution and 0.4 mL of 0.025 M phosphate buffer (pH = 11).

Donor atom type

The donor atom type is one of the most important factors in determining the 99mTc-labeling efficiency. The rank order for the four thiol-containing chelators is in HaL4 > H3L3 > H4LI > H4L2. The labeling conditions required for H4LI and H4L2 are roughly comparable. Under the same conditions, H3L3 gives higher radiolabeling yield. H3L4 is well labeled with RCP > 9 0 0 under milder conditions (at pH 7 and room temperature) at very low concentration (1.25 ×

10 5 M, ~ 5/~g mL-~). Apparently, the substitution of amide-nitrogen by amine-nitrogen enhances the ligand exchange rate. This is consistent with the results from kinetic studies of various thiol-protected MAMA (monoamide-monoaminedithiol) chelators using gluconate as the exchange ligand (Rao et al., 1992). H4L2 reacts with [99=Tc]glucoheptonate complex and forms the expected complex [99mTcO(L2)] under alkaline (pH > 9.0) conditions. At lower pH (pH < 7.0) several hydrophilic ~gmTc species with

2.5 x 10 -4 M

1.25 x 10-3 M

h.

t-

k._

i . 2 5 x 10 -5 M

2.5 x 10 -5 M

I' I

5

,

l

l

L

I

I

I0

15

0

5

10

Time (Minutes)

I

15

|

20

Time (Minutes)

Fig. 2. Radio-HPLC chromatograms for complexes [~TcO(L2)]-(left side, method I) and [9~TcO(L4)]

(right side, method 2) at concentrations of 2.5 x 10 -4 M (top) and 1.25 x 10 -5 M (bottom).

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retention times of 9-11 min (left side, Fig. 2) are formed in addition to the expected complex [99mTcO(L2)] at a retention time of 12 min. The amount of these 9OmTc species decreases at lower chelator concentration or using a longer heating time. At higher pH, these hydrophilic 99mTCspecies can be easily converted to the complex [99mTcO(L2)]-. Similar characteristics were reported for the S-benzoyl-protected MAG3 when used in the exchange labeling (Bormans et al., 1995). The more hydrophilic ~gmTc species is probably a kind of precomplex in which the Tc atom is coordinated by more than one thiolate-S donors from the H4L2 ligand molecules (Bormans et al., 1995). Since the amide-nitrogen atoms become deprotonated at higher pH, the chelator is able to bind the Tc as a tetradentate ligand. As a result, the precomplex is converted to ~gmTcO(Ll)] (Fig. 3). Similar behavior was observed for H,L2, but not for H~L3 and H3L4. Chelators H,LI-H3L3 form technetium complexes (Fig. 3) with two isomeric forms (cis- and trans-) because of the relative orientation of the functional group to the T ~ O bond. The HPLC chromatograms of these three complexes showed two radiometric peaks (left side, Fig. 2) corresponding to the two isomeric forms. The complex [TcO(L4)] has only one isomeric form, and its HPLC chromatogram (right side, Fig. 2) showed one radiometric peak.

99mTc-labeling o f H Y P Y

Unlike tetradentate thiol-containing chelators (H4LI-H3L4), HYPY bonds to the Tc only as a mono- or bidentate ligand. Thus, the choice of the coligand is critical. Abrams and coworkers have shown that a Tc(V) oxo intermediate is required for successful radiolabeling, and compounds with a' putative Tc(O)O4 structure, e.g. [99mTc]glucoheptonate (Kieviev, 198 l), [99mTc]saccharate (Pak et al., 1988) and [~gmTc]-2-hydroxymethylbutyric acid (Colombo et al., 1988) react with HYN1C-derivatized molecules. In this study, we choose glucoheptonate as the coligand because [99mTcO(GH)z]- was used as an intermediate complex for ligand exchange with the four thiol-containing chelators. [~mTcO(GH)2] has also been used as an intermediate to label hydazino-modified proteins with ~mTc (Abrams et al., 1990; Schwartz et al., 1991). The reaction of HYPY with [~mTc]glucoheptonate was carried out by heating the reaction mixture at 50~C for 30 min under acidic conditions (pH 4-5). The concentration of glucoheptonate was 20 mg mL '. The HYPY concentration ranged from 2.5 x 10 -6 to 2.5 x I 0 - 4 M . The % RCP was caculated using the sum of peaks at retention time of 5-9 min (Fig. 4). The labeling yields at various concentrations are summarized in Table 4. In all the

COOH

(CRy3 HOOC

[Wm'rco(, ~)T

['QmTcO(L1)]"

HOOC,L

EK:X)C .,COOe ~wr NI'I,~/N'~I/

ON i~1~ H

[mnTCO(L4)]

[~"ml'cOlL3)]

! N

O

. : o"V,', ' '~'~,....J ~ " " O OH OH HO._~% H H [~"Tc{HYPY)(GH)2] Fig. 3. ~mTc complexes.

Radiolabeling efficiencyof chelators

I

I

I

I

0

5

10

15

I

20

Time (Minutes) Fig. 4. Radio-HPLC chromatogram (method 1) for the complex [~mTc(HYPY)(GH)2]. cases, [99mTc]colloid formation was minimum. It is clear that the higher HYPY concentration produces better RCPs for the complex [99mTc(HYPY)(GH)2]. Several radiometric peaks at retention times of 5-9 min were observed for the complex [99mTc(HYPY)(GH)2] in its HPLC chromatogram (Fig. 4), suggesting multiple 99mTc species in the reaction mixture. We attribute the presence of the multiple species to the resolution of some of many possible isomers that can result from different bonding modalities of HYPY and the two glucoheptonate coligands. The formation of multiple species was also seen in the complexes [~gmTc(HYPY)(tricine)2] and [99mTc(HYNICtide)(tricine)2], where tricine is the coligand and the HYNICtide (cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6hydrazinonicotinamido)hexanamide)))) is a HYNICderivatized cyclic platelet glycoprotein lib/Ilia receptor antagonist (Liu et al., 1996a). The retention times for different 99mTc-labeled hydrazines vary systematically with their lipophilicity, while the peak pattern in the radio-HPLC chromatograms remains relatively unchanged. The ratios of the peak areas in the radio-HPLC chromatogram (Fig. 4) change with time, reaction temperature, and pH. This is probably caused by the conversion from a kinetically favored mixture of coordination isomers to a more thermodynamically favored mixture of coordination isomers. In order to prove this assumption, we have isolated several fractions of the peaks at 5-9 min and collected them into a solution of excess glucoheptoTable 4. Effect of H Y P Y concentration on radtolabeling yield Concentration (M) 2.5x10 4 1.25xl0 -4 2.5x10 -~ 1.25x10-5 2.5xl0 -6

Average RCP (%) 96.1 95.6 90.2 77.0 65.2

+__ 1.0 _+ 0.8 + 1.4 _+ 6.2 _ 5.3

1109

nate. In the absence of glucoheptonate, the collected fractions were not stable enough for further characterization. Reinjection of a single fraction produces most other peaks at 5-9 min. This strongly suggests that the formation of the multiple species is, in fact, due to coordination isomerism and not due to the formation of radioimpurities or artifacts of the chromatographic conditions. The exact nature of the bonding between the Tc center and HYPY, as well as the glucoheptonate coligand, remains unclear. Complexes containing Tc-hydrazido and Tc-diazenido bonds have been previously reported and characterized by X-ray crystallography (Nicholson et al., 1989; Archer et al., 1990: Cook et al., 1990; Abrams et al., 1991; Dilworth et al., 1994). If one assumes that the coordination geometry around the Tc is distorted octahedral and HYPY binds to the Tc by forming a Tc-hydrazido bond, it requires two glucoheptonate (GH) ligands to form the complex [99mTc(HYPY)(GH)2] (Fig. 3). In conclusion, the relative 9~mTc-labeling efficiency of several potentially tetradentate thiol-containing chelators was studied by competing them with glucoheptonate in the [99mTc]glucoheptonate complex. There are several factors that influence the labeling efficiency of a chelator. These include donor atoms, chelator concentration and reaction conditions such as the temperature and reaction time, and the pH of the reaction media. In all the cases, higher chelator concentration produces better radiolabeling yields. Heating at 100:C for 30 min is required for the successful 99mTc-labeling of N2S2 diamidedithol (H4LI) and N~S triamidethiol (H4L2), while the N2S2 monoamide monoaminedithiol (H3L3) can be well labeled under milder conditions. For the N2S2 diaminedithiol (H3L4), the ligand exchange could be completed within 60 min at room temperature. It is clear that substitution of an amine-N for an amide-N enhances the rate of 9~mTc-labeling. The radiolabeling efficiency of four thiolcontaining chelators was also compared to that of 2-hydrazinopyridine, a model compound for HYNIC. The radiolabeling efficiency of HYPY using glucoheptonate as coligand is better than that of H4LI-H4L2, is slightly better than that of H3L3, and is comparable to that of H3L4 under conditions used in this study. Therefore, HYNIC and diaminedithiols are the candidates of choice as bifunctional coupling agents in labeling small biologically active molecules with very high potency. It should be noted that the 99mTc-labeling efficency of a bifunctional coupling agent is also dependent on the identity of the exchange ligand (or coligand) for both thiol-containing chelators and aromatic hydrazines. The use of the appropriate conbination of bifunctional coupling agent and exchange ligand (or coligand) can still result in 99mTc-labeling biologically active molecules with high specific activity.

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Acknowledgements--The authors acknowledge Dr M. Rajopadhye and J.P. Bourque for the synthesis of chelators, 4,5-bis[S-benzoyl(mercaptoacetamido)]pentanoic acid and 2-[(S-trityl-mercapto)ethylaminoacetyl]-S-trityl-L-cysteine ethyl ester, and to K. Yu for the synthesis of N,N'ethylenediyl-bis-L-cysteine diethyl ester (H3L4).

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