Bioconjugate Chem. 1993, 4, 78-84
78
Radioiodination of Proteins Using N-Succinimidyl 4 - Hydr oxy-3-iodobenzoate Ganesan Vaidyanathan, Donna J. Affleck, and Michael R. Zalutsky' Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710. Received September 23, 1992
N-Succinimidyl4-hydroxy-3[13111iodobenzoate([13111SHIB)was synthesized from 4-hydroxybenzoic acid in two steps. The overall radiochemical yield was 40-56%. A monoclonal antibody (mAb) was labeled in 10-15% yield by reaction with [l3lI1SHIB. The specific binding of [13lI]SHIB mAb to tumor homogenates in vivo was 78 f 3 % ,compared to 84 f 3% for the same mAb labeled using N-succinimidyl 3- [l25IIiodobenzoate ( [l25I1SIB). Paired-label studies in normal mice demonstrated similar tissue distributions of 1311and 1251 except in thyroid. In thyroid, uptake of the two isotopes was similar on day 1; however, 1311levels increased gradually to 2-3 times those of 1251by day 6. Our results indicate that loss of label in vivo from mAbs labeled using SHIB is somewhat higher than seen with SIB but significantly lower than that observed when direct iodination methods are used.
INTRODUCTION
Monoclonal antibodies (mAbsl) and other proteins labeled with isotopes such as 1311,1251, 12*1,and 1231 have been utilized in a wide variety of basic and clinical investigations (1-3). In these studies, the method most commonly used for a protein radioiodination is direct iodination using an oxidant such as Iodogen or Chloramine-T (4, 5 ) , generally resulting in the formation of iodinated tyrosine residues (Chart I). Proteins labeled by direct iodination are ideal for most in vitro applications; however, proteins labeled by this approach frequently undergo extensive deiodination in vivo (6). Loss of label is reflected by accumulation of radioiodine in thyroid and stomach and with mAbs, a diminished pool of activity available for tumor uptake (7). Since direct iodination results in the formation of iodotyrosine residues on the protein, we hypothesized that the action of deiodinases involved in thyroid hormone metabolism (8)might be responsible for the deiodination of mAbs in vivo (9). In order to circumvent the action of these enzymes, we developed an approach for labeling mAbs using the acylation agent N-succinimidyl 3-iodobenzoate (SIB), a reagent synthesized via the electrophilic destannylation of N-succinimidyl3- (tri-n-butylstanny1)benzoate(9).This method involves coupling to lysine residues and, unlike iodotyrosine, does not result in the substitution of the iodine ortho to a hydroxylgroup on an aromatic ring (Chart I). Paired-label studies demonstrated that mAbs and F(ab')2 fragments labeled using SIB exhibited 40-100fold lower levels of thyroid uptake, reflecting a greater inertness to dehalogenation compared to mAbs labeled directly (10, 11). Similar results have been obtained by Wilbur et al. (12)using the para isomer of SIB. The current study is directed at refining our understanding of the structural requirements needed for a protein iodination site in order to inhibit deiodination in
* Address correspondence to Michael R. Zalutsky, Ph.D., Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710. FAX: (919)684-4211. Abbreviations used: mAbs, monoclonal antibodies; SHIB, N-succinimidyl4-hydroxy-3-iodobenzoate; HIBA, 4-hydroxy-3iodobenzoic acid; SIB, N-succinimidyl 3-iodobenzoate; IBA, 3-iodobenzoic acid.
Chart I. Structure and Attachment Site of Iodinated Moieties of Radioiodinated Proteins: Direct vs Conjugation Methods ~H2CHzCONH(CH,).
bH Direct
SIB
bH Bolton-Huntei
SHIB
vivo, particularly with regard to the role played by the o-hydroxyl group. In a previous report (131,we evaluated the utility of the Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy-3-iodophenyl)propionate, for in vivo applications of labeled mAbs. Even though labeling proteins via the Bolton-Hunter reagent involves substitution of the radioiodine ortho to a hydroxyl group (Chart I), pairedlabel experiments demonstrated that mAbs labeled using the Bolton-Hunter method exhibited 1order of magnitude lower deiodination than directly labeled mAb and a thyroid uptake only twice that observed for mAb labeled using SIB. In addition to the presence or absence of the o-hydroxylgroup, the iodination site on mAbs labeled using SIB and the Bolton-Hunter agent differ with regard to another structural factor: SIBlacks the two-carbon spacer between the aromatic ring and the active ester moiety. To determine whether this two-carbon spacer was an important factor in reducing dehalogenation, we embarked on this study evaluating the utility of N-succinimidyl4-h~droxy-3-iodobenzoate (SHIB)for mAb labeling. As shown
1043- 18Q2/93/29Q4-QQ78~Q4.QQ~Q 0 1993 Amerlcan Chemical Society
BloconJu~~t8 Chem., Vol. 4, No. 1, 1993 79
Protein Radlolodlnatlon
in Chart I, SHIB differs from SIB only in the presence of the o-hydroxyl group. The secondary motivation of this study was more pragmatic in nature. If the SHIB reagent yielded a labeled protein resistant to deiodination in vivo, this labeling method would obviate the need for the synthesis and purification of an N-succinimidyl (trialkylstanny1)benzoate precursor and only commercially available compounds would be required. Our results indicate that mAbs can be labeled with SHIB with retention of immunoreactivityand that loss of label in vivo is somewhat higher than seen with SIB but considerably lower than that observed with direct iodination methods.
ethanol (38% yield): mp 170-173 "C. [lit. mp 175-176 "C (17);169-173 "C (IS)].HPLC and TLC demonstrated the presence of 4-hydroxybenzoic acid; however, this did not interfere with the next step. The HIBA (254 mg, 1.0 mmol) was dissolved in 3 mL of dry THF, and 150 mg (1.3 mmol) of N-hydroxysuccinimide and 204 mg (1mmol) dicyclohexylcarbodiimide were added. The mixture was stirred overnight under argon at room temperature. The precipitated dicyclohexylureawas filtered and the precipitate was washed with THF. The combined filtrate was evaporated, yielding 362 mg of an off-white solid. This was purified by silica gel chromatography using ethyl acetate as the solvent to yield EXPERIMENTAL PROCEDURES 120 mg (34%) of a white solid: mp 189-191 "C; TLC (EtOAc)Rf= 0.69; IR (KBr) cm-l3242,1756,1719; NMR General. NMR spectra were obtained in appropriate (CD3CN) 6 1.93 (m, residual protons from the solvent;), solvents with a General Electric Midfield GN-300 spec2.82 (s,4 H), 7.02 (d, 1 H, J = 9 Hz), 7.80 (m, 1H), 8.44 trometer. For organic solvents, the proton chemicalshifts (d, 1H, J = 24 Hz); MS m/z (FAB) 362,247,138,121,107, were reported in ppm downfield from internal TMS (0.00 93,77; HRMS calcd for CllHgIN05 (M + H+) 361.9525, ppm);where NaOD solution was used, the HOD peak (4.75 found 361.9534. ppm) was used as the reference. No chemical shift Preparation of Glycine Conjugateof 3-Iodobenzoic reference was used for carbon-13 NMR. IR spectra were Acid. N-Succinimidyl 3-iodobenzoate (prepared from obtained on a BOMEM MB-100 variable-resolution FTIR 3-iodobenzoic acid and N-hydroxysuccinimide; 139 mg, spectrophotometer. Mass spectral data were obtained on 0.4 mmol) in 2.5 mL of THF was added to glycine (30.2 a VG-705 (VG Analytical, Danvers, MA) or on a Hewlettmg, 0.4 mmol) in 1.25 mL of 0.1 M borate buffer, pH = Packard GC/MS/DS Model HP-5988A instrument. Melt8.5. After stirring for 2 h a t room temperature, the hazy ing points were determined on a Haake Buchler variablereaction mixture turned homogenous. Since TLC (25% heat apparatus or on a Fisher Johns melting point MeOH/EtOAc) performed 1h later showed the presence apparatus and are uncorrected. of unreacted ester, 50-60 mg of glycine was added to the All reagents were of reagent grade or better. The tin above mixture with periodic addition of buffer and THF precursor for SIB preparation, N-succinimidyl 3-(tri-nto keep the reaction mixture homogenous. After two days butylstannyl) benzoate, was prepared and radioiodinated of stirring at room temperature, the reaction mixture was using previously described procedures (14). made alkaline with 1 N NaOH and the unreacted ester Sodium [125I]iodide and sodium [l3lI1iodide were obwas extracted into ethyl acetate. The aqueous layer was tained from Du Pont-New England Nuclear (North acidified and the resultant white crystalline precipitate Bellerica, MA) in 0.1 N NaOH solution. Monoclonal was extracted with ethyl acetate. After washing with brine, antibody 81C6 is of the IgGzb isotype and reacts with an the ethyl acetate layer was dried with sodium sulfate and epitope of the extracellular matrix antigen tenascin (15). evaporated to give 67 mg (557% ) of a white solid mp 154This mAb was obtained as a gift from Dr. Dare11 Bigner, 156 "C [lit. mp 156.3 "C (19)l; IR (KBr) cm-l3280,1741, Department of Pathology, Duke University Medical 1620,1549,1201; NMR (NaOD) 6 4.75 (HOD), 3.87 (s,2 Center. H), 7.21 (t, 1 H, J = 8 Hz), 7.70 (d, 1 H, J = 8 Hz), 7.85 Thin-layer chromatography was performed on EM (d, 1H, J = 8 Hz), 8.09 (s, 1H); 13CNMR-APT (NaOD) Science analytical silica plates. Flash chromatography 631.92 (CH2),81.85 (CI), 114.31 (CH), 118.34 (CH), 122.99 was done with 230-400 mesh silicagel from VWR scientific, (CCO), 123.94 (CH), 156.20 (CONH), 164.46 (COOH). Marietta, GA. High-pressure liquid chromatography Preparation of Glycine Conjugate of 4-Hydroxy(HPLC) was conducted with two systems. For standards 3-iodobenzoicAcid. This compound was prepared in an and lipophilicity measurements, a Perkin-Elmer series 4 identical fashion as above. Starting with 274 mg (0.74 liquid chromatograph was used. The peaks were detected mmol) of SHIB, 330 mg of crude product was obtained. with Perkin-Elmer LC-95 UV/visible spectrophotometer A portion (200 mg) of this was purified by preparative detector. For radioiodinated compounds,HPLC was done TLC using 5050 ethyl acetate/methanol to yield 141 mg with two LKB Model 2150 pumps, an LKB Model 2152 of a glassy solid mp 88-90 "C; TLC (50:50 ethyl acetate/ control system, an LKB Model 2138 fixed-wavelengthUV methanol) Rf= 0.38; IR (KBr) cm-l 3629, 3545, 3139 (v detector, and a Beckman Model 170radioisotope detector. br), 1730,1645,1572,1500,1407,1214,764;NMR (NaOD) With this system, peak analysis was performed with a 6 4.75 (HOD), 3.09 (8, 2 H), 6.40 (d, 1H, J = 8.6 Hz), 7.37 Nelson Analytical software package on an IBM computer. (dd, 1 H, J = 2.5 and 8.6 Hz), 7.97 (d, 1 H, J = 2.5 Hz); An Alltech 250 X 4.6 mm Adsorbosphere 10-micron C18 l3C NMR-APT (NaOD) 6 32.13 (CHZ),79.66 $11, 105.45 column and an Alltech 250 X 4.6 mm partisil 10-micron (CH), 107.67 (CCO), 117.67 (CH), 126.59 (CH), 157.27 column were used for reverse-phase and normal-phase (COH), 158.54 (CONH), 165.64 (COOH); MS m/z (CI) HPLC, respectively. Depending on the activity level, 339 (M NH4+), 322 (M + H+), 281,264, 220,196,180, radioactivity was measured with a Capintec CRC-7 ra138;HRMS (FAB)calcd for CgHgIN04 (M + H+)321.9576, dioisotope calibrator or with a LKB 1282 dual-channel y found 321.9575. counter. Preparation of Radiolabeled 4-Hydroxy-34odobenPreparation of N-Succinimidyl4-Hydroxy-3-iodozoic Acid and Its NHS Ester. To a solution of benzoate. 4-Hydroxybenzoic acid was iodinated following 4-hydroxybenzoic acid (5 pL; 3.8 mg in 4 mL of 0.05 N a literature procedure (16) for the preparation of 3-(4'NaOH) was added 1-2 pL of 1311(or lZ5I)in 0.1 N NaOH hydroxy-3'-iodopheny1)propanoic acid. Starting from 3.3 (0.2-1.0 mCi) followed by 5 pL of chloramine-T solution g (24 mmol) of 4-hydroxybenzoic acid, 5.6 g (89% ) of crude (10 mg/mL in 0.1 M borate, pH = 8.5). After 10-min 4-hydroxy-3-iodobenzoic acid (HIBA) was obtained. The reaction at room temperature, 7 pL of sodium bisulfite (40 product was purified by crystallizing twice from 50%
+
Vaidyanathan et al.
00 Bioconjugate Chem., Vol. 4, No. 1, 1993
mg/mL in borate buffer) was added. The reaction mixture was injected onto a HPLC ((218, 110:89:1 MeOH/H20/ HOAc; 0.5 mL/min). Retention time for 4-hydroxybenzoic acid was 9.8 min and that for the iodinated product was 15.8 min. More than 80% of the injected activity eluted with the retention time of [1311]HIBA. A small amount of a coelutingimpurity was seen in the UV profile;however, by excluding the first 1mL of the peak, [l3lI1HIBA could be isolated from this impurity. Most of the methanol from the HPLC fraction containing the ['3lI]HIBA was evaporated with an argon stream, and the activity in the residual aqueous solution was extracted into ethyl acetate (3 X 500 pL). The ethyl acetate solution was dried with sodium sulfate and evaporated to about 100pL, transferred to a l/Z-dram vial, and evaporated to dryness. N-Hydroxysuccinimide and dicyclohexylcarbodiimide(3pmol in each of 30pL of THF) were added to the above activity, and the mixture was left at room temperature for 2-3 h. The reaction mixture was injected onto HPLC (silica) and eluted with 50:502 (v/ v/v) ethyl acetate/hexane/acetic acid at 1mL/min. The product ( t =~ 10.8 min) was isolated in 50-70% radiochemical yield. Coupling of [ l311lSHIB to 81C6. Most of the solvent from the HPLC fraction containing [13111SHIB was evaporated with argon and transferred to a llz-dram vial and the solvent was further evaporated to dryness. To the above activity was added a solution (75pL, 5 mg/mL) of mAb 81C6 in borate buffer (0.1 M, pH = 9.0). The mixture was incubated at room temperature for 1h. The reaction was terminated by the addition of 0.3 mL of 0.2 M glycine. The labeled mAb was purified by passage through a Sephadex G-25 column and eluted with phosphate-buffered saline (PBS). The protein-associated activity was measured by TCA precipitation and compared to that of 81C6 labeled using [lZ5I]SIBin a paired-label fashion. Trichloroacetic acid precipitable activity was 98.9 f 0.3% and 99.7 f 0.1% for 81C6 labeled using [l3lI]SHIB and [lz5I1SIB,respectively. In Vitro Specific Binding Measurements. About 100 ng each of 81C6 labeled via [lz5I1SIBand [l3lI]SHIB were added in triplicate to 250 mg of both antigen-positive D-54 MG human glioma and antigen-negative rat liver homogenates. The homogenates were incubated overnight at 4 "C, washed three times, and counted for 1311and 1251 activity. Specific binding in this single point assay was defined as the percentage of activity bound to tumor minus the percentage bound to liver. Tissue Distribution Measurements. These studies were performed in paired-label format in normal male BALB/c mice weighing 22-30 g. Groups of five or six animals were injected in the tail vein with a mixture of both preparations and sacrificed by ether overdose at selected time points. Tissues of interest were removed, washed with saline, and weighed. A dual-channel y counter was used to determine tissue activity levels of 1311and 1251. The percent injected dose per organ was calculated by comparison to standards of appropriate count rate. The first set of experiments compared the tissue distribution of [l3lI]HIBA and [125I]IBA, compounds which are potential labeled catabolites of mAbs labeled using [l3lI1SHIBand [l25I]SIB, respectively. [l3lI]HIBA was prepared as described above and its purity was greater than 99% as confirmed by TLC. To prepare [1251]IBA, [125IlSIB was incubated with 1 mL of 1 N NaOH for 1 h at room temperature. This solution was acidified with 250-300 pL of 6 N HC1. The activity was extracted into ethyl acetate (3 X 500 pL). The ethyl acetate solution was
washed with brine and dried with sodium sulfate, and the ethyl acetate was evaporated. Little or no activity was lost in this step. The dried activity was reconstituted in PBS. TLC of hydrolyzed products was run using 1% acetic acid in ethyl acetate as the eluent. More than97 % coeluted with an authentic IBA standard and 0.50% was retained at the origin. Mice were injected with 7 pCi of [l3lI1HIBA and 5 pCi of [125111BAand groups of animals were sacrificed at 30 min and 1 and 2 h. In a similar study, blood and urine samples were obtained 1and 2 h after injection of [l3lI1HIBAand [lz5I]IBA for HPLC analysis. In each case, samples were pooled from three to five animals. Urine samples were diluted with phosphate-buffered saline and passed through a 0.2pm syringe filter (Acrodisc, Gelman Sciences). Blood samples were centrifuged, and sera were passed through a 10-kD cutoff Centricon (Amicon, Danvers, MA). Samples were analyzed using a reverse-phase HPLC system described in a previous publication (20). The second set of paired-label biodistribution measurements were performed to compare the distribution of radioactivity following the administration of mAb radioiodinated using SHIB and SIB. Animals were injected via the tail vein with 8 pg (3 pCi) of 81C6 labeled with [l3lI1SHIBand 3 pg (4 pCi) of 81C6 labeled with [1251]SIB. Groups of animals were studied at 1, 2, 5,6, and 7 days after injection. Lipophilicity Measurement. The relative lipophilicities of the two potential catabolites were measured by a reverse-phase HPLC system (21). The retention times of the two compounds were determined by using an Alltech Adsorbosphere C18 10-micron column and 59.4:39.6:1 (v/ v/v) H20/MeOH/HOAc as the eluent at a flow rate of 1 mL/min. The capacity factor k' was calculated for each compound and was used as an indicator of relative lipophilicity. Statistical Analysis. Since the tissue-distribution experiments were performed in a paired-label format, with each animal serving as ita own control, a paired t test was used to compare the biodistribution of the two radioiodinated mAbs as well as that of the potential catabolites. RESULTS AND DISCUSSION
When evaluating potential methods for the radioiodination of mAbs, a number of factors must be considered. First, the mAb should be labeled in reasonable radiochemical yield with retention of antigen-binding capacity. Second, the bond between the radioiodine and the mAb should be stable in vivo. Third, the labeled species generated by the catabolism of the mAb should be excreted rapidly. The potential utility of SHIB as an acylation agent for labeling mAbs was evaluated using the antiglioma mAb 81C6 IgG because of the availability of data for this mAb using a variety of radioiodination methods (11,13,20). A potential advantage of SHIB compared to SIB is that due to the presence of the hydroxyl group, iodination via electrophilic substitution should proceed in reasonable yield. Thus, SHIB can be labeled from commercially available compounds and the synthesis, and purification of an N-succinimidyl (trialkylstanny1)benzoateprecursor is not required. Initially, the labeling of SHIB in one step by reaction of N-succinimidyl 4-hydroxybenzoate with chloramine-?' and l31I was attempted. Since only modest yields were obtained and separation of SHIB from unreacted radioiodine and excess starting ester was problematic, the two-step procedure outlined in Scheme I was adopted.
Protein Radioidination
Bloconjugate Chem.. Voi. 4, No. 1, 1993 81
Scheme 1. Synthesis of N-Succinimidyl 4-Hydroxy-3[1311]iodobenzoate
IBA 0 HIBA I
FOOH
ChloramineT bH
VI bH
)Ajl bH SHIB
In the first step, 4-hydro~y-3-[13~I]iodobenzoic acid was prepared by reaction of sodium [13111iodidewith 4-hydroxybenzoic acid using chloramine-T as the oxidant. Yields of more than 80% were obtained and the product could be purified using reverse-phase HPLC. A 2-3-h reaction of the labeled acid with N-hydroxysuccinimide and dicyclohexylcarbodiimide gave the desired product, [13111SHIB,in 50-70 % yield. The [13'I]SHIB acylation agent was purified using normal-phase HPLC prior to reaction with mAlj. An important advantage of SIB compared to the BoltonHunter reagent is that its mAb coupling efficiencyis about 2-fold higher (12). This difference was considered to be related to the higher rate of competitive hydrolysis imparted by the two-carbon spacer between the ester moiety and the aromatic ring present in the Bolton-Hunter reagent (Chart I). Since SHIB also lacks this spacer, it might be expected that protein coupling should proceed in good yield with this reagent. However, even with a reaction time of 60 min and a relatively high pH (9.0)and protein concentration (5 mg/mL), the coupling efficiency of SHIB to mAb 81C6 was only 10-15 % ;even poorer yields were obtained with shorter reaction times. In comparison, after only a 15-20-min reaction, more than 70% of SIB could be coupled to mAb 81C6 and yields for the BoltonHunter reagent were 2530% (13). We believe that the low mAb coupling efficiency for SHIB can be attributed to the presence of the hydroxyl group para to the ester. Unlike the case with the BoltonHunter reagent, this p-hydroxyl group enhances the resonance stabilization of the ground state, thereby reducing the rate of nucleophilic attack at the carboxyl carbon (22). This ground-state stabilization is also reflected by the fact that HIBA is a weaker acid than IBA (23).
A potential advantage of SHIB for labeling mAbs is
that like other conjugation methods, direct exposure of the mAb to oxidants is avoided. Using asingle-point assay, the specific binding in vitro of mAb 81C6 labeled using [131I]SHIB was 78 f 3 % , a value comparable to that observed in parallel studies with mAb labeled using [l25IISIB (84 f 3 %). The specific binding percentages for mAb labeled with both acylation agents are significantly higher than those reported previously for the same mAb labeled with l3lI using Iodogen (24). The tissue distribution of HIBA and IBA were compared in normal mice in order to evaluate the effect of the hydroxyl group on the tissue distribution of radioiodine. In addition, the distribution of these labeled acids are of interest because they are likely catabolites of mAbs labeled using SHIB and SIB. Because of the proclivity of iodide for the thyroid, uptake of activity in this tissue is used frequently as an indicator of deiodination. As shown in Figure 1, thyroid uptake was quite low after injection for both HIBA and SIB. Thyroid accumulation of HIBA increased from 0.13 f 0.02% injected dose at 30 min to
a
El
1,
STOMACHI
14
INTESTINES~
1.
0.
0.1
TIME (HOURS)
0.5
1
2
Figure 1. Comparative tissue uptake in mice following the coadministration of [131]HIBA and [1251]IBA. BLOOD 1H
10
HIBA
HIBA
I
Y
Iodide I
5
--
I
v
HIBA-Gly I I
v
8
'1
Iodide
IBA
I
04 0
5
10
15
20
25
1
I
30
Fraction Number
Figure 2. Reverse-phaseHPLC chromatogram of blood sample obtained 1 h after coadministrationof [l3lI1HIBAand [125111BA.
0.26 f 0.07% at 2 h. In comparison, thyroid uptake of IBA was significantly lower ( P < 0.005),ranging from 0.03 f 0.01% to 0.05 f U.M% over the same time period. Although these results suggest 4-5-fold greater dehalogenation of HIBA compared to IBA, it should be noted
82
Vaidyanathan et al.
Bloconjugate Cham., Vol. 4, No. 1, 1993 URINE 1H
10
HIBA
I
I
0
10
5
0
15
20
25
30
F'raction Number
Figure 3. Reverse-phase HPLC chromatogram of urine obtained 1h after coadministration of [1311]HIBA and [lZ5I]IBA. SHIB
m
0
0.5
a
9 bp
1
2 5 6 Time (Days)
7
Figure 4. Thyroid uptake in mice followingthe coadministration of 81C6 mAb labeled with lS1I using [l3lI]SHIB and with lZ5I using [I2511SIB. Statistically significant differences ( P < 0.01) indicated by asterisk.
that subtle but indistinguishable levels of free iodide in the two preparations (both about 0.5-0.9% ) could also account for the 0 . 1 4 2 % differences in thyroid uptake of HIBA and IBA. Although metabolic cages were not used to insure complete urine collection, data obtained for activity in
urine and bladder at death indicate that excretion of HIBA is significantly greater than for IBA (Figure 1). For example, 1 h after injection, bladder and urine activity was 60.1 f 13.1% for HIBA, a value significantly higher than that observed for IBA (40.9 f 3.1 % ,P < 0.01). The more rapid urinary excretion of HIBA may be related to ita lower lipophilicity. When the lipophilicities of HIBA and IBA were measured by reverse-phase HPLC, the k' value for HIBA was 3.31 compared to 4.59 for IBA. Liver uptake of HIBA was about 5 times higher than that of IBA at all time points, an observation that might appear to be discordant with the lower lipophilicity of HIBA. However, higher liver selectivity for hydrophilic compounds has been reported (25) and the metabolism of HIBA may differ from that of IBA due to the presence of the hydroxyl group (see below). In general, the tissue distribution observed for IBA is in good agreement with previously published results (IO), with the exception that somewhat lower activity levels were seen in blood in the current study. This may be related to the more stringent conditionsused to hydrolyze SIB to IBA. If small amounts of unreacted SIB were present due to incomplete hydrolysis, labeling of serum proteins in vivo might be possible, since incubation of mouse serum with incompletely hydrolyzed SIB resulted in protein labeling in vitro (data not shown). Previous studies have shown that mAbs labeled by reaction with SIB are excreted via the urine primarily as glycine conjugates (20). However, due to the resonance stabilization of SHIB as hypothesized earlier, formation of HIBA-glycine conjugates might not be favorable. One hour after injection, conversion of IBA to ita glycine conjugate was observed in blood (Figure 2) and urine (Figure 3) while the HIBA-glycine conjugate was not seen in these same animals. About 40-45% of the HIBA was present in urine as an unknown metabolite with an elution time that indicated that it was more hydrophilic than the HIBA-glycine conjugate. This unidentified compound was not seen in blood. The HPLC pattern at 2 h was similar for both blood and urine except the amount of free iodide and IBA-glycine conjugate in urine was roughly equal. Excretion of aromatic acids via glycine conjugate formation has been shown to depend on a number of factors including species (261,dose, and any coadministered substances (27). In the case of salicyclic acid, benzoate (28) and p-aminobenzoic acid (29) have suppressed the formation of glycine conjugates. In rabbits, 0-(salicyclic acid) and m-hydroxybenzoic acids were eliminated essentially unchanged (30);however, about 33% of the para isomer was eliminated as the glycine conjugate (31).
Table I. Paired-Label Tissue Distribution of Radioiodine in Normal Mice following Injection of 81C6 Labeled with [l29]SIB and with [1311]SHIB % injected dose per organa 1day 5 days 6 days organ SIB SHIB SIB SHIB SIB SHIB liver 5.16f 1.01 4.88f 0.97 1.85 f 0.36 2.12 f 0.37 1.16 & 0.34 1.40 f 0.44 0.32 f 0.04 0.14f 0.02 0.14 f 0.02 0.36 f 0.04 0.10 f 0.01 0.11 f 0.01 spleen 2.59 f 0.81 1.64 f 0.48 2.69 f 0.83 0.45 f 0.07 lungs 1.95 f 0.60 0.55 0.07 heart 0.53 f 0.06 0.27 f 0.08 0.57 f 0.06 0.24 f 0.07 0.13f 0.05 0.16 f 0.06 kidneys 0.86f 0.10 2.18 f 0.25 2.37 f 0.29 1.16 f 0.10 0.57 0.14 0.87 f 0.18 stomach 0.22 f 0.06 0.15 f 0.06 0.18 f 0.07 0.61f 0.08 0.56f 0.08 0.25 f 0.07 1.35 f 0.20 0.76f 0.23 am int 3.26 f 0.12 3.07f 0.10 1.15 f 0.19 0.62f 0.18 lg int 1.44 f 0.13 0.61 f 0.10 0.75f 0.13 0.42 f 0.16 1.41 f 0.12 0.31 f 0.10 muscle 13.85f 1.65 12.57 f 1.68 6.24 f 0.76 4.79 f 0.47 6.84 f 0.84 5.60 f 0.56 blood 28.65 f 4.26 29.14 f 4.49 12.54f 1.63 6.26 f 2.22 10.24 f 1.49 8.08f 3.02 brain 0.15 f 0.02 0.07 f 0.01 0.05 f 0.02 0.15f 0.02 0.07f 0.01 0.04f 0.01 Mean f SD (n = 5 ) .
*
*
Protein Radlolodinatlon
Identification of the unknown peak found in urine has not been attempted at this time. This species could be the glucuronide or sulfate, other common metabolites of aromatic acids (27). Paired-label studies were performed in normal mice to compare the in vivo distribution of radioiodine from mAb 81C6 IgG labeled by reaction with [l3lI1SHIBand [12511SIB. In thyroid, uptake of 1311(Figure 4) suggested that, at early time points, the presence of the hydroxyl group does not increase deiodination. However, with time thyroid uptake of 1311becomes 1.7-2.9 times higher than 1251with these differences considered to be statistically significant (P< 0.01) on days 5-7. As summarizedin Table I, the tissue distribution of both nuclides was quite similar in other tissues. At later time points, retention of radioiodine from mAb appears to be somewhat higher in kidney (P< 0.01, days 5 and 6) and several other tissues when labeling was performed using SHIB. Although these differences are small, and generally not significant, these trends are not inconsistent with the differences observed in the in vivo behavior of the potential catabolites HIBA and IBA. The thyroid uptake levels determined in this study for 81C6 labeled using SIB are in excellent agreement with those obtained previously with this mAb in normal mice (13,20)but again are 2-3-fold higher than those observed in athymic mice (11). This underscores the importance in evaluating various labeling methods in paired-label format in order to eliminate effects related to species and metabolic and nutritional status. The results of this study can be evaluated most effectively by comparison to other paired-label experiments performed with mAb 81C6, and several conclusions regarding the effect of iodination site on deiodination can be made. SHIB lacks a two-carbon spacer between the aromatic ring and the active ester and the Bolton-Hunter reagent contains this spacer, yet both exhibited about 2-fold higher thyroid uptake than SIB (13). Although this two-carbon spacer increases the structural difference between the labeling site and that found on mAbs labeled on tyrosine residues, it does not appear to influence the deiodination of mAbs labeled using the Bolton-Hunter reagent. Thyroid uptake of HIBA was only 0.13-0.26 7% of injected dose and that of its mAb conjugate was generally less than 0.5%. In comparison, when mAb 81C6 was labeled using Iodogen, 1order of magnitude higher thyroid uptake was seen (13).This suggests that the deiodinases thought to be responsible for the deiodination of tyrosine residues on directly labeled mAbs are enzymes with a high degree of specificity. This is supported by the fact that other molecules containing a hydroxyl group ortho to the site of iodination also exhibit relatively low accumulation in thyroid (32-34). Nonetheless, it is important to note that the thyroid uptake of IBA and its mAb conjugate are somewhat lower than observed for HIBA and its mAb conjugate. Deiodination levels are quite low with both acylation agents, possibly reflecting the fact that deiodinases do not play a major role in the loss of label from either compound. In that case, differences in the stability of HIBA and IBA might be due to the lower carbon-iodine bond strength imparted by the hydroxyl group (35). In conclusion, although SIB appears to remain the reagent of choice for labeling mAbs, the presence or absence of a hydroxyl group ortho to the iodination site is not the only factor to be considered in the design of mAb radioiodination methods.
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ACKNOWLEDGMENT
The excellent technical assistance of Susan Slade is greatly appreciated. The authors are grateful to Sandra Gatling for help in preparing this manuscript. This research was supported by National Institutes of Health Grants CA 42324, NS 20023, CA 14236, and by Grant DEFG05-89ER60789 from the Department of Energy. LITERATURE CITED (1) Bolton, A. E., and Hunter, W. M. (1973)The labelling of
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