Nitriles form mixed-coligand complexes with 99mTc-HYNIC-Peptide

Nuclear Medicine and Biology 29 (2002) 107–113

Nitriles form mixed-coligand complexes with

99m

Tc-HYNIC-Peptide

Guozheng Liua, Charles Wescottb, Aaron Satob, Yi Wanga, Ning Liua, Yu-Min Zhanga, Mary Rusckowskia,*, Donald J Hnatowicha a

Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655, USA b Dyax Corp., Cambridge, MA, 02139 USA Received 11 April 2001; received in revised form 25 May 2001; accepted 18 August 2001

Abstract Using a 12-amino acid peptide conjugated with HYNIC as a model, we investigated nitriles as possible coligands for labeling with 99mTc. After the preparation of the 99mTc labeled HYNIC-peptide using tricine as coligand, the addition of acetonitile was found by reverse phase HPLC to block further coligand exchange with ethylenediamine diacetic acid (EDDA) at room temperature. The addition of this nitrile changed the pharmacokinetics of the 99mTc labeled peptide in normal mice towards faster clearance and significant differences in accumulation in most tissues sampled. By replacing acetonitrile with cyanoacetate, a nitrile not present in the HPLC eluant, it was possible to show the existence of a new, more hydrophilic, species by reverse phase HPLC. We conclude that nitriles can act as coligands for HYNIC-conjugated peptides labeled with 99mTc and tricine. Furthermore, the presence of acetonitrile during Sep-Pak or HPLC purification may inadvertently generate a mixed tricine/acetonitile coligand 99mTc-HYNIC-peptide complex. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Peptide; HYNIC; Tricine; Acetonitrile;

99m

Tc

1. Introduction The bifunctional chelator, NHS-hydrazinonicotinamide (NHS-HYNIC), is widely used for labeling peptides and proteins with 99mTc [1]. Since HYNIC alone cannot satisfy the coordination requirements of Tc(V), coligands such as glucoheptonate [1,5–10,19,27,28], tricine [11–16,21,23,25– 28], ethylenediamine diacetic acid (EDDA) [16,17,23], tricine/phosphine [18,20,25], and tricine/pyridine [16,17, 24] have been used. The complex of 99mTc-HYNIC-peptide with coligands EDDA [23], tricine/phosphine [18,20,25], and tricine/pyridine [16,17,24] can all be prepared by coligand exchange. The EDDA complexes can also be prepared by labeling the HYNIC-peptide directly with EDDA as coligand, but the labeling efficiency is much lower [16,17]. While attempting coligand exchange of tricine with EDDA by first immobilizing the preformed 99mTc-HYNIC-peptide/tricine complex on a C18 Sep-Pak cartridge, washed first with an EDDA solution and then eluted with 80% acetonitrile, no exchange * Corresponding author. Tel.: ⫹1–508-856 – 6972; fax: ⫹1–508-856 – 4572. E-mail address: [email protected] (M. Rusckowski).

was apparent by reverse phase HPLC. This suggested that acetonitrile in the mobile phase may have blocked the exchange by acting as a coligand itself. This possibility is reasonable, since HYNIC-peptide complexes with the mixed coligands tricine/phosphine and tricine/pyridine have been made [18,24] and nitriles are also monodentate neutral ligands similar to phosphines and pyridines. The chemistry of technetium complexes with TcO3⫹ and TcN2⫹ cores is well understood. The complexes have a square pyramid structure with Tc⫽O or Tc⬅N at the apical position while the four basal positions are occupied by various ligands. The Tc⫽N-NH-3⫹ core is very similar to the TcO3⫹ and TcN2⫹ in complex formation and structural geometry [22]. The structure of several Tc⫽N-NH-3⫹ compounds have been determined by x-ray single crystal diffraction and shown to be also square pyramid [2– 4]. These Tc(V) cores combine with various coordinating atoms, but if neutral, the coordinating atoms cannot occupy all four positions, otherwise the coordination sphere will then bear a high positive charge. After coligand exchange between tricine in 99mTc-HYNIC-peptide complexes and phosphine or pyridine, Edwards et al determined that only one basal position is occupied by phosphine or pyridine [18,24].

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If acetonitrile can incorporate as a coligand into the 99m Tc-HYNIC-peptide/tricine complex, then depending upon the kinetics of formation, HPLC analysis with acetonitrile eluants may generate mixed tricine/acetonitrtile coligand complexes on the column not present in the sample under analysis. For the same reason, if acetonitrile is used in a Sep-Pak purification of 99mTc-HYNIC-peptide/tricine preparations, a mixed tricine/acetonitrtile coligand complex may inadvertently result. Finally, if acetonitrile and other nitriles can participate in the complex, the properties of the 99m Tc-HYNIC-peptide may be influenced. It has been shown that replacing the coligand can strongly influence the biodistribution of the radiolabel [6,16]. Here we report on the use of nitriles (acetonitrile and cyanoacetate) as coligands for 99mTc-HYNIC-peptides and we show that these nitriles have a significant effect on the in vivo properties of the 99mTc radiolabel.

2. Materials and methods The 12-amino acid peptide (DX180) was a gift from Dyax Corp (Cambridge, MA). The peptide was obtained already conjugated with HYNIC via a terminal carboxyl group and a hexanoic acid linker. The HYNIC conjugated peptide was received as a 1.1 mM solution in DMF with a peptide purity of 99% as determined by mass spectrometery and HPLC analysis. All other chemicals were purchased and used without purification. 99mTc-pertechnetate was obtained from a 99Mo-99mTc radionuclide generator (Dupont, Billerica, MA). 2.1. HPLC and paper chromatography Reverse phase HPLC was performed on a C18 column (YMC-pack, ODS-AMQ, S-5 ␮m, 25 ⫻ 0.46 cm, Waters, Milford, MA) using a Waters Millenium system with an in-line radioactivity detector. The gradient system was run at a flow rate of 1 mL/min using the following conditions: Eluant A consisted of 0.01 M Phosphate Buffer (pH 6.2); and eluant B was 100% acetonitrile. For the first 5 min the system was run at 10% B, increasing over 5– 8 min to 30% B, then over 8 –25 min to 37% B, then over 25–30 min to 60% B, then returning to 10% B in 2 min and finally remaining at 10% B for 8 min. Paper chromatography was performed on Whatman No. 1 paper (Whatman International Ltd, Maidstone, England ) with acetone as eluant for detection of 99mTc-pertechnetate (Rf⫽1.0), and 40% acetonitrile-water solution for determination of 99mTc colloid (Rf ⫽ 0.0). The Rf values for the labeled peptides in these two systems were 0.0 and 1.0 respectively. Each 10 cm paper strip was cut into 10 pieces for counting in a NaI (Tl) gamma counter.

2.2. Preparation of coligand

99m

Tc-HYNIC-peptide with tricine as

To prepare the 99mTc-labeled peptide with tricine as coligand, 5 ␮L of the HYNIC-peptide DMF solution was added to 25 ␮L of 0.25 M ammonium acetate buffer, pH 5.2, 10 ␮L of 0.56 M tricine, and followed with 2– 6 ␮L of 99m TcO4- eluate (about 2 mCi). After mixing, 3 ␮L of a fresh solution of SnCl2 䡠 2H2O (1.11 mM, in 10 mM HCl containing 5.05 mM sodium ascorbate) was added. After 1 h incubation at room temperature, an additional 3 ␮L of the tin solution was added (at a higher concentration of 4.44 mM, in 10 mM HCl, containing 5.05 mM sodium ascorbate) to prevent air oxidation of the 99mTc complex. Samples were analyzed by C18 reverse phase HPLC and paper chromatography. 2.3. Coligand exchange with EDDA with and without added acetonitrile To 5 ␮L of the preformed 99mTc-HYNIC-peptide/tricine preparation was added 0.1 mL of an EDDA solution (10 mg/mL, pH 7.0). The solution was incubated for 2 h at room temperature and then heated in a boiling water bath for an additional 20 min. To test the effect of aecetonitrile on this exchange, 5 ␮L of acetonitrile was added to the 5 ␮L of preformed 99mTc-HYNIC-peptide/tricine preparation 30 min before the addition of EDDA. Samples were analyzed by HPLC as before. 2.4. Preparation of 99mTc-HYNIC-peptide with tricine and cyanoacetate as coligands To an aliquot of 10 ␮L of the 99mTc-HYNIC-peptide/ tricine preparation was added 20 ␮L of a cyanoacetate aqueous solution (1.88 M, pH 5.0) followed by incubation for 1 h at room temperature. 2.5. Preparation of 99mTc-HYNIC-peptide/tricine/acetonitrile and 99mTc-HYNIC-peptide/tricine/cyanoacetate mixture The mixed preparation method of Edwards et al [18] was used to determine the coordination number of nitriles in the 99m Tc-HYNIC-peptide complex. To a 10 ␮L aliquot of the labeled 99mTc-HYNIC-peptide/tricine solution was added 20 ␮L of a 1.88 M aqueous cyanoacetate solution and 20 ␮L of a 1.88 M aqueous acetonitrile solution (i.e. 10% acetonitrile to match the initial concentration used in the HPLC analysis). The solution was incubated for 1 h at room temperature. The samples were then analyzed by HPLC. 2.6. Preparation of 99mTc-HYNIC-peptide with tricine and acetonitrile as coligands The 99mTc-HYNIC-peptide with tricine/acetonitrile as mixed coligands was prepared using the following coligand

Liu et al. / Nuclear Medicine and Biology 29 (2002) 107–113

109

exchange method. A C18 Sep-Pak cartridge was preconditioned with 10 mL ethanol followed by 10 mL water. A sample of 99mTc-HYNIC-peptide with tricine as coligand was applied to the column followed by 5 mL of a tricine solution (pH 5.2) containing 11.2 mM tricine, 0.013 M ammonium acetate, and 0.133 mM SnCl2 䡠 2H2O. The labeled peptide was then eluted with 5 mL of 80% acetonitrile in the tricine solution described above. Fractions were collected and those containing the peak radioactivity were dried under a stream of nitrogen and reconstituted in the pH 5.2 tricine solution to a concentration of about 350 ␮Ci/mL. 2.7. Biodistribution in mice of 99mTc-HYNIC-peptide with tricine and tricine/acetonitrile as coligands The 99mTc-HYNIC-peptide with tricine and tricine/acetronitrile as coligands were prepared as described above and then diluted to 350 ␮Ci/mL with the pH 5.2 tricine solution mentioned above for administration. All animal studies were performed with the approval of the Institutional Animal Care and Use Committee. Normal CD-1 male mice (Charles River, Wilmington, MA) were injected through a tail vein with 1 nmole (35 ␮Ci) of either labeled peptide. Mice were anesthetized with metofane (Schering-Plough, Omaha, NE) and sacrificed by heart puncture at 0.5, 1, and 3 h. Organs of interest were removed and counted in a NaI(Tl) automatic gamma well counter along with a sample of the injectate. The remaining carcass was counted in a dose calibrator after the contents of the bladder were carefully removed.

3. Results The labeling efficiency for 99mTc-HYNIC-peptide/tricine was 95% (2% s.d., N⫽18) as determined by both HPLC and paper chromatography, with recovery of radioactivity on HPLC typically greater than 85%. 3.1. Acetonitrile can block EDDA coligand exchange with tricine Figure 1 presents HPLC radiochromotograms of 99mTcHYNIC-peptide under different conditions. Panel A is that of the tricine preparation showing one prominent peak. Panel B is that of the preparation after 2 h incubation at room temperature with EDDA without the addition of acetonitrile. Several additional peaks appear before that of the main peak. Panel C is that of the identical preparation, also after 2 h of incubation at room temperature with EDDA but in the presence of acetonitrile added 30 min before (see Methods). The presence of added acetonitrile has consolidated the radiochromatogram to that approaching the initial tricine preparation. Panels D and E present identical radiochromatograms generated by heating at 100 °C for 20 min the preparations shown in panels B and C respectively.

Fig. 1. Reverse phase HPLC radiochromatograms of 99mTc-HYNIC-peptide with tricine (panel A), after incubation of 99mTc-HYNIC-peptide/ tricine with EDDA at room temperature for 2 h without (panel B) and with (panel C) the addition of acetonitrile. The figure also shows radiochromatograms of 99mTc-HYNIC-peptide/tricine/EDDA preparation (i.e. panel B) after heating to 100 °C (panel D) and 99mTc-HYNIC-peptide/tricine/ EDDA/acetonitrile preparation (i.e. panel C) after heating to 100 °C (panel E).

The fact that incubation of 99mTc-HYNIC-peptide/tricine complex with EDDA for 2 h at room temperature results in several new smaller peaks (Figure 1B), demonstrates that coligand exchange can occur although the exchange is very inefficient for EDDA under these conditions. The addition of acetonitrile to the 99mTc-HYNIC-peptide/tricine complex 30 min before the EDDA blocks the formation of these intermediate species (Figure 1C). That EDDA coligand exchange can be prevented by acetonitrile suggests that the complex referred to here as 99mTc-HYNIC-peptide/tricine/ acetonitrile has been formed with acetonitrile. If the kinetics

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Liu et al. / Nuclear Medicine and Biology 29 (2002) 107–113

of acetonitrile incorporation into the complex is sufficiently rapid, the acetonitrile complex may form during loading of 99m Tc-HYNIC-peptide/tricine on the HPLC column for analysis. This seems to be the case, because the prominent peak in Figure 1A for 99mTc-HYNIC-peptide/tricine has the same retention time as that of 99mTc-HYNIC-peptide/ tricine/acetonitrile in Figure 1C and may therefore represent the same complex. Other small peaks located between 16 and 24 min in Figures 1 A and B must be due to labeled peptide species since they appear and disappear only by the addition or subtraction of EDDA and acetonitrile. To our knowledge, all published methods for the reverse phase HPLC analysis of 99mTc-labeled peptides include acetonitrile in the eluant. Our attempts to achieve satisfactory resolution without acetonitrile were unsuccessful. Consistent with reports by others [16,17,23], heating a mixture of 99mTc-HYNIC-peptide/tricine in the presence of EDDA results in the nearly complete formation (about 95%) of the 99mTc-HYNIC-peptide/EDDA complex as shown in Figure 1D. That this complex has a short retention time, 17 min, indicates that the EDDA complex is more hydrophilic than the mixed coligand tricine/acetonitrile complex. After heating the mixture of EDDA and 99mTc-HYNIC-peptide/ tricine/acetonitrile complex (Figure 1C), the EDDA complex is again obtained as shown by the similarity of radiochromatograms Figures 1 D and E. The presence of acetonitrile is unable to block the formation of the EDDA complex when heated. 3.2. Cyanoacetate as coligand Since acetonitrile is present in the reverse phase HPLC eluant, its presence cannot be avoided in providing direct evidence that acetonitrile is not involved in 99mTc-HYNICpeptide complex formation. Accordingly, cyanoacetate was selected as an alternative nitrile. Because one methyl hydrogen in acetonitrile is replaced by a carboxylate group, the substitution of acetonitrile with cyanoacetate should render the complex more hydrophilic and therefore eluting earlier on HPLC (however this requires that the cyanoacetate complex be sufficiently inert kinetically to avoid exchange with acetonitrile during analysis). Figure 2 presents the radiochromatogram of 99mTcHYNIC-peptide/tricine with cyanoacetate (Figure 2B) along with that of 99mTc-HYNIC-peptide/tricine (Figure 2A) for comparison. As shown, the addition of cyanoacetate results in a new species eluting early at 16.7 min and therefore more hydrophilic than 99mTc-HYNIC-peptide/tricine/acetonitrile. The small peak at 21 min in Figure 2B may be due to incomplete exchange. 3.3. Coordination number Figure 2C presents the radiochromatogram of the exchange mixture of 99mTc-HYNIC-peptide/tricine with both acetonitrile and cyanoacetate. According to Edwards, et al

Fig. 2. Reverse phase HPLC radiochromatograms of 99mTc-HYNIC-peptide/tricine (panel A), 99mTc-HYNIC-peptide/tricine after incubation with cyanoacetate (panel B) and 99mTc-HYNIC-peptide/tricine after incubation with a mixture of acetonitrile and cyanoacetate (Panel C).

[18,24], if one basal position in the 99mTc-HYNIC-peptide/ tricine complex is occupied by another coligand, then there should be two peaks in the radiochromatogram. In the case of 99mTc-HYNIC-peptide/tricine with both cyanoacetate and acetonitrile, one peak will be due to the tricine/cyanoacetate complex and the other to the tricine/acetonitrile complex. If two basal positions are occupied, then there should be three peaks corresponding in this case to the 99m Tc-HYNIC-peptide/tricine complex with two cyanoacetates, two acetonitriles, and one cyanoacetate and one acetonitrile. As shown in the figure, only two peaks are evident suggesting that the nitriles are only occupying one basal coordination site. Therefore the remaining basal positions must still be occupied by tricine leading to a mixed coligand complex. 3.4. Biodistribution in mice Biodistribution results can also provide evidence for a new coligand complex. Table 1 lists the tissue radioactivity levels at three time points following administration of 99m Tc-HYNIC-peptide/tricine and 99mTc-HYNIC-peptide/ tricine/acetonitrile to normal mice. As shown, the presence of acetonitrile in the preparation procedure has resulted in significantly less radiolabel accumulating in the whole body at all time points (p⬍0.01) and in most tissues at each time

Liu et al. / Nuclear Medicine and Biology 29 (2002) 107–113 Table 1 Biodistribution in mice of value by Student’s t-test Organ

99m

99m

0.5 h

Tc-HYNIC-peptide/tricine/NCCH3. Values shown are percent ID/organ. (N ⫽ 4). p

1.0 h

Tricine

Liver Heart Kidneys Lung Spleen Stomach Sm. Int. Lg. Int. Blood Whole body*

Tc-HYNIC-peptide/tricine and

Tricine/NCCH3

Mean

s.d.

Mean

s.d.

8.24 0.24 4.06 0.63 0.21 1.64 2.49 0.80 7.43 67.09

1.13 0.04 0.58 0.05 0.07 0.61 0.35 0.18 1.95 4.14

6.30 0.14 4.26 0.35 0.15 0.72 5.01 0.45 5.14 46.19

1.10 0.02 1.56 0.04 0.04 0.09 0.87 0.03 1.24 4.32

p

0.039 0.007 0.800 0.000 0.176 0.056 0.001 0.027 0.100 0.000

111

3.0 h

Tricine

Tricine/NCCH3

Mean

s.d.

Mean

s.d.

7.58 0.21 3.51 0.51 0.19 2.24 2.72 1.20 6.36 60.70

0.35 0.03 0.37 0.07 0.01 0.42 0.11 0.09 0.63 4.47

5.17 0.12 2.63 0.24 0.11 0.36 4.03 0.58 3.08 40.5

0.43 0.01 0.60 0.02 0.03 0.07 0.49 0.16 0.17 1.61

p

0.000 0.002 0.054 0.000 0.010 0.000 0.011 0.001 0.172 0.000

Tricine

Tricine/NCCH3

Mean

s.d.

Mean

s.d.

5.12 0.13 2.51 0.34 0.13 0.92 1.99 2.53 3.71 45.45

0.47 0.02 0.18 0.04 0.01 0.09 0.22 0.55 0.37 0.61

3.36 0.09 2.39 0.20 0.10 0.24 1.19 3.84 2.06 30.73

0.73 0.01 0.37 0.03 0.03 0.06 0.33 1.08 0.21 2.52

p

0.009 0.006 0.556 0.000 0.207 0.000 0.009 0.087 0.000 0.001

* Whole body radioactivity is the sum of radioactivity in each organ and carcass minus urine radioactivity. p values ⱕ 0.05 bolded.

point. Radioactivity levels are significantly ( p⬍0.05) higher for 99mTc-HYNIC-peptide/tricine/aceotnitrile only in the small intestines. For all other tissues sampled, when differences are significant, values are higher for 99mTcHYNIC-peptide/tricine.

4. Discussion That acetonitrile in the presence of 99mTc-HYNIC-peptide/tricine will form the mixed-coligand complex 99mTcHYNIC-peptide/tricine/acetonitrile may be viewed as reasonable. It has been reported that phosphine and pyridine derivatives will form mixed coligand complexes with 99m Tc-HYNIC-peptide/tricine [18,24]. In common with phosphines and pyridines, nitriles are soft monodentate ligands with neutral coordinating atoms. According to the structure proposed by Edwards, et al, for tricine/phosphine and tricine/pyridine mixed coligand complexes [18,24], the proposed structure of 99mTc-HYNIC-peptide/tricine/nitrile should be that shown in Figure 3. As discussed above, the coligand competition exchange study between acetonitrile and cyanoacetate also indicates that only one nitrile is in the coordination sphere.

Fig. 3. Proposed structure for the mixed coligand complex of [18,24] for tricine and phosphine or pyridine.

99m

The labeling of bioactive molecules with 99mTc using HYNIC follows a mechanism quite different from that of alternative approaches using N2S2 and N3S chelators. In the later case, the chelators themselves provide the four basal atoms. The HYNIC technetium link Tc⫽N-NH-3⫹, like Tc⫽O3⫹ or Tc⬅N2⫹, often behaves like a core with regard to exchange reactions. The common features of these cores are multiple bonds between technetium and nitrogen or oxygen, short bond length (1.56 –1.63 Å), and an oxidation state of ⴙ5 for technetium. To form a complex, the core will combine with four other coordinating atoms forming a square pyramid structure with the core at the apical position [22]. There is occasionally a weakly bonded atom at the sixth position trans to the core. The four atoms at the basal positions are required to bear some negative charge to neutralize the positive charges and to form some ␲ character bonds to further delocalize the electron cloud on the technetium. Sometimes these two requirements are not satisfied by the same ligands, leading to the formation of mixed coligand complexes. In this report we did not study the kinetics of the coligand exchange between nitriles and the tricine coligand complex. If the peak observed by HPLC of 99mTc-HYNIC-peptide/

Tc-HYNIC-peptide with tricine and nitrile compared to that proposed by Edwards et al

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Liu et al. / Nuclear Medicine and Biology 29 (2002) 107–113

tricine corresponds to a 99mTc-HYNIC-peptide/tricine/acetonitrile complex, then the reaction is very fast at room temperature since one sharp peak is prevalent (Figure 1A). The relatively high radioactivity levels in stomach listed in Table 1 for 99mTc-HYNIC-peptide/tricine compared to 99m Tc-HYNIC-peptide/tricine/acetonitrile are probably due to 99mTc-pertechnetate formed in vivo in the case of the former complex. We have recently shown that the 99mTcHYNIC-peptide/tricine is unstable to air oxidation and that incorporation of acetonitrile renders the complex much more stable against oxidation (data not presented). The 99m Tc-HYNIC-peptide/tricine complex could also be oxidized similarly in vivo. An explanation for the higher whole body and tissue radioactivity levels observed for 99mTcHYNIC-peptide/tricine may be related to the tendency of this complex to bind to serum proteins (data not presented). Should the mixed tricine/acetonitrile complex be more stable towards protein binding, the results would be expected to be faster clearance and lower tissue radioactivity levels.

5. Conclusions Nitriles as neutral monodentate compounds, together with tricine can form mixed-coligand complexes with 99m Tc-HYNIC-peptides. When reverse phase HPLC with acetonitrile in the eluant is used for analysis of these complexes, the possibility that mixed complexes may be formed during the analysis should be considered. For the same reason, if a 99mTc-HYNIC-peptide/tricine complex is prepared using Sep-Pak and acetonitrile, the purified product may include the mixed coligand complex. Finally, the mixed coligand complexes of 99mTc-HYNIC conjugated peptides with tricine/nitriles may be considered a new class of mixed coligand complex, possibly with unique and useful in vivo properties.

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