Nucl. Med. Biol. Vol. 19, No. 8, pp. 897-902, 1992 Int. J. Radial. Appl. Instrum. Part B Printed m Great Britain. All rights reserved

0883-2897/92 SW0 + 0.00 Copyright 0 1992 Pergamon Press Ltd

Photoreactive ‘I1In-Cyclodextrin Inclusion Complex: a New Heterobifunctional Reagent for Antibody Labeling THEODORE

S. T. WANG, RONALD

Division

RASHID

L. VAN

A. FAWWAZ

and

HEERTUM

of Nuclear Medicine, Department of Radiology, Columbia University, New York, NY 10032, U.S.A. (Received

12 March 1992)

The compound of interest, N-5-azido-2-nitrobenzoylaminomethyl-”’In-acetylacetone-a-cyclodextrin (CD) (V) was synthesized by the selective tosylation of a-CD to form Gtosyl-6-deoxy-CD, which was then reacted with NaN, to form 6-azido-6-deoxy-CD (11).This was followed by catalytic hydrogenation to yield III. Compound III and “‘In-acetylacetone were mixed to form an inclusion complex, which was then reacted with N-5-azido-2nitrobenzoyloxysuccinimide to yield compound V. Anti-melanoma MAbTP41.2 was added to compound V, followed by immediate photoreactivation labeling by U.V.light at 320 nm. The final product M was purified from a Sephadex G-50 column. “‘In-DTPA-MAbTP41.2 was also prepared as a control. Immunoreactivity via the cell-binding assay of VI was 87%, compared with 57% by the BADTPA method. Biodistribution in non-tumor rats yielded a liver concentration in %ID/g of 3.5, 1.7 and 1.O for compound VI, compared to the 5.5, 5.2 and 3.1 for the BADTPA compound, at 4, 24 and 48 h post-injection, respectively.

Introduction

Materials and Methods

c(-, /3- and y -cyclodextrins (CDs) are cyclic oligosacchat-ides consisting of 6, 7 and 8 glucose units. They are toxicologically harmless compounds which can be obtained by enzymatic degradation of starch (Szejtli, 1984) and form a wide variety of inclusion compounds with molecules that fit into their 5-8 A cavities (Saenger, 1980). As opposed to the classical clathrate compounds, these inclusion complexes can exist in aqueous solution. In a previous communication (Wang et al., 1991) we have reported briefly that a-CD can form an (“’ Ininclusion complex with ‘I’ In-acetylacetone ACAC). Based on this finding, N-5-azido-Znitrobenzoylamidomethyl-c -CD (V), a photoreactive heterobifunctional reagent, was designed and synthesized. This reagent can form an inclusion complex with I” In-ACAC. In addition, its aryl azide terminus can insert into a C-H bond, and hence is capable of

All reagents were purchased from commercial suppliers and used as received, unless otherwise specified. MAbTP41.2 is an IgG2a that recognizes a human high molecular weight melanoma-associated antigen. Chemical reagents used are abbreviated as follows:

reacting with any amino acid residue of a monoclonal antibody (MAb). In this paper, we have examined the

use of this new photoreactive heterobifunctional reagent as compared with the currently most commonly used bicyclic anhydride of DTPA (BADTPA) with regard to immunoreactivity and animal biodistribution.

ACAC = acetylacetone CL -CD = o(-cyclodextrin ANB-NOS = N-5-azido-Z nitrobenzoyloxysuccinimide BADTPA = bicyclic anhydride of diethylenetriaminepentaacetic acid DMSO = dimethyl sulfoxide. Melting points were determined on a Fisher-Johns apparatus and are uncorrected. Thin-layer chromatography (TLC) and instant thin-layer chromatography (ITLC) were performed on Silica gel G with the following solvents: A, 14: 3 : 3 butanone-methanolwater; B, 12 : 3 : 5 butanone-methanol-l M acetic acid; C, normal saline solution; D, 1:l chloroform-ethyl acetate. Iodine vapor and 10% aqueous sulfuric acid spray followed by heating were used to detect the cyclodex-

trin derivatives on the TLC plates. 897

THEODORE S. T. WANG ef al.

898

CH2-0

CH,OH

~

;g;=e

~

-.,:IH$jJj-N3

(1)

a-Cyclodextrin

WI

CH2 -

NH2

CH2 -

NH,

N, N3&-COO-b

0 N-5-AzidwZ-nitrobenzoyloxysuccinimide (ANS-NOS) O.lM PBS, pH 5.0

CH2 -

NHCO

NH-MAbTP41.2

MAbTP41.2

(W U.V. light 320-350 nm RT 5min (photolysis)

“‘In-ACAC-a-C&2nitrobenzoyl-MAbTP41.2

Fig. 1. Synthesis of “‘In-ACAC-a-CD-2-nitrobenzoylaminomethyl-MAbTP41.2

(VI).

Photorcactive “I In-cyclodextrin inclusion complex Preparation of 6-0-p-tolylsulfonyl-a-CD

(Z) (Fig. 1)

a-CD (4.86 8, 5 PM) and p-toluenesulfonyl chloride (19 g, 0.1 mM) were dissolved in dry pyridine (SOOmL). The reaction mixture was stirred at room temperature for 1 h, and the reaction stopped by adding 50 mL of water. Pyridine was removed in vacua until a syrup was obtained. Water (150 mL) was added to this product, and the evaporation was continued. This process was repeated several times until no odor of pyridine was detectable. The aqueous solution (4OOmL) was filtered and applied to a column of Darco, G-60,2040 mesh. The column was eluted with water, followed by 30% ethanol and 25% 1-propanol. The I-propanol eluate from the column was collected in 100 mL fractions. Concentrated ammonia (2 mL) was added to each fraction, and the solutions were evaporated to dryness. The product was crystallized from 95% ethanol and yielded 2.18 g (39%), m.p. 157-159°C (literature 159-162°C) (Croft and Bartsch, 1983; Melton and Slesser, 1971). The purity of the compound was assayed by TLC using solvent A. Preparation of 6-azido -6-deoxy -a -CD (ZZ)

Compound I (1.13 g, 1 PM) and sodium azide (1.3 g, 20 mM) in water (100 mL) were heated at 100°C under reflux for 1 h. The reaction mixture was cooled to room temperature, filtered and then evaporated in vacua to a low volume (c. 4mL). The product was washed with an equal amount of 1,1,2,2tetrachlorethane (4 mL). The tetrachlorethane layer was separated from the aqueous solution by centrifugation. Evaporation of the aqueous solution yielded the product (II), which was further purified by crystallization from water. The yield was 0.5Og (49%). TLC (solvent B) indicated that the product was pure, m.p. 215°C (literature 217°C) (Croft and Bartsch, 1983; Melton and Slesser, 1971). Preparation of 6-amino -6-akoxy -a -CD (ZZZ)

Compound II (0.49 g, 0.5 PM) was dissolved in water (100 mL), and palladium black (200 mg) was added at 14 lb/in* of hydrogen in a Parr hydrogenator at room temperature overnight. After the catalyst was filtered, the solution was evaporated in vacua to dryness. The product was obtained by crystallization from water and yielded 0.44 g (91%), m.p. 196199°C (literature 200°C) (Croft and Bartsch, 1983; Melton and Slesser, 1971). TLC (solvent B) indicated a pure product. Preparation of “‘In -ACAC 1.5 mCi/O.lS mL of 0.04 M HCl, of “‘In chloride was added to acetylacetone (2Opg, 0.2 pmol) in 0.2 mL HEPES solution, at pH 8.0. The mixture was vortexed briefly and kept at room temperature for 1Omin. The reaction mixture was then extracted with 3 mL (3 x ) of chloroform. The chloroform layer was separated, combined and evaporated. The residue was dissolved in 0.5 mL of ethanol-normal

899

saline (1: l/v/v) solution. The purity of the product was identified with ITLC (chloroform+thyl acetate 1: 1, v/v), (R,= 1.0) and Whatman No. 1 paper (n-butanol-n-propanol-H,O, 9 : 6 : 3, v/v/v) (R,= 0.55-0.65). Preparation of “‘In-ACAC-6-amino-6-deoxy-a-CD (ZV)

The preparation of IV was previously described (Wang et al., 1991). Briefly, “I In-ACAC (1.2 mCi) in 1 mL of ethanol-normal saline solution was added to compound III (0.29 mg, 0.3 ,uM) in 0.1 M PBS, pH 7.4. The reaction mixture was kept at room temperature for 2 h, then at 4°C overnight. ITLC (solvent C and D) was used to monitor the yield of IV. The product was purified by adding a large amount of absolute ethanol. The precipitates were collected by centrifugation, and washed with absolute ethanol. Finally the precipitates were dissolved in 0.1 M PBS, pH 8.0 (1 mL). The purity of the product was checked again by ITLC using solvents C and D, which indicated a pure product (Croft et al., 1983; Melton et al., 1971). Compound IV was immediately used for the preparation of compound V as described below. Preparation of N-Sazido-2+itrobenzoylaminomethyl-“‘In-ACAC-a-CD (V)

ANB-NOS (0.3 mg, 1 PM) in 30 PL of DMSO was added to compound IV (0.9 mCi) in 1 mL of 0.1 M PBS, pH 8.0. The mixture was kept at room temperature for 30 min and immediately used for the preparation of compound VI as described below. Preparation of “I In -ACAC-a -CD-2-nitrobenzoylaminomethyl-MAbTP41.2 (VZ) (Imai et al., 1990; Lewis et al., 1977)

Anti-melanoma MAbTP41.2 (4 mg, 2 x 10e5 mmol) dissolved in 0.1 M PBS, pH 7.4 (2 mL) and V were mixed at room temperature under continuous stirring. Ultraviolet irradiation was carried out in a U.V. light generated from a bank of two FS-20 Westinghouse lamps which emitted a flux of 1 mW/cm* at 320nm as determined by a u.v.-X radiometer (Ultraviolet Products, San Gabriel, Calif.), for 1Omin at a rate of 10 J/m’/s u.v.-B light at a distance of 10cm. The product was purified on a Sephadex G-50 column and eluted with 0.1 M PBS, pH 7.4. The MAB peak fractions were combined. The concentration of MAB was assayed by the Bio-Rad protein assay method (Bradford et al., 1976) and the absorbance was determined by a spectrophotometer at 595 nm. A serial of known amount of bovine y-globulin was used as standard. Preparation of “‘In-DTPA-MABTP41.2 control study

(VtZ) for

Compound VII was prepared by a conventional method (Szejtli, 1982). Briefly, BADTPA (21 pg) was mixed with MAbTP41.2 (4 mg) in 1 mL of 0.1 M

THEODORE S. T. WANG et al.

900

PBS, pH 8.6, at room temperature for 1 h. The reaction mixture was dialyzed in 0.1 M PBS, pH 7.4, at 4°C overnight. “‘In-acetate then was added at pH4.0, and the mixture was incubated at room temperature for 1 h. The product was isolated by passage through a Sephadex G-50 column, and eluted with 0.1 M PBS, pH 7.4. The product was further purified by extensive dialysis at 4°C against 0.1 M PBS, pH 7.4. In vitro stability studies

Stability of compound (VI) was tested in vitro by mixing 100 p L (100 pg) of (VI) with 2 mL of human serum albumin. The mixture was put in a sterile vial and placed in an incubator (37°C) filled wih an air/CO, mixture (95/S%). Twenty five PL aliquots of the solution were sampled out at 4, 8 and 24 h of incubation and analyzed by an electrophoresis (7.50% polyacrylamide gel) system. Five ,uL of the samples were applied on the slab gel. Protein markers were identified by Coomassie Blue stain. The gel was frozen, cut into 3 mm sections and assayed for radioactivity in a y-counter. Immunoreactivity assays

Melanoma cells (human COLO-38, 1 x 106) or B-lymphoblastoid cells (human Wil-2, 1 x 106) were incubated with VI and VII in a 96-well polyvinyl chloride microliter plate. After 1 h of incubation, the cells were washed 3 times and cell-bound was determined in triplicate in the region of antigen excess (plateau region).

NOS. After photoaffinity labeling of MAbTP41.2 with V, the final compound VI was isolated from the Sephadex G-50 column and eluted with 0.1 M PBS, pH 7.4. The radiochemical yield of “‘In-ACAC was 82% at 1.23 mCi/O.l6~mol ACAC. The yield of compound (IV) was 74% at 0.91 mCi/O.l2pmol. Compound (IV) was immediately used to prepare (V) with ANB-NOS. After photoaffinity labeling of MABTP41.2 with (V), the final compound (VI) was isolated from a Sephadex G-50 column, and yielded 0.59 mCi/3 mg MABTP41.2. Labeling efficiency was 66%. Specific activity was 0.2 mCi/mg MABTP41.2. Immunoreactivity assay

Direct cell binding immunoreactivity assays revealed that at the plateau region, 87.1 & 8.4% of VI and 57.8 _+6.5% of VII were bound to melanoma cells (P < 0.001). Only 3.1 & 0.4% of VI and VII were bound to lymphoma cells. Animal studies

The results of biodistribution in non-tumor rats for “‘In-ACAC, “I In-ACAC-a-CD, VI and VII are summarized in Table 2(a) and (b). At 48 h post-injection, the blood concentration of VI was relatively lower than that of VII, and the liver concentration of VI was significantly lower (1.04%) (P < O.OOl), than that of VII (3.13%). The kidney concentration was higher for VI as compared with VII.

Discussion As in starch, a-CD contains glucose units linked by

Animal studies

a-( l-4) bonds. The chair forms of the six-membered

Compound VI and a control radiolabeled compound [“‘In-DTPA-MAbTP41.2 (VII)] were injected into the tail vein of non-tumor rats (Lewis), n = 3-5 per group. The animals were sacrificed at 4, 24 and 48 h post-injection. Tissue was removed, weighed and the radioactivity determined in a well-type scintillation counter. Statistical analyses were performed using an unpaired Student’s test. Results are presented as mean + SD.

glucose rings are all aligned in register (Szejtli, 1982). Because of the apparent lack of free rotation about the glycoside bond which connects the glucose units, the CDs are not perfectly cylindrical molecules but are somewhat cone shaped (Rebek, 1988; Saenger, 1984a). The 6-hydroxyl face is the narrow side while the 2,3-hydroxyl face is somewhat wider. The important structural features to notice in CDs are their toroidal shape, hydrophobic cavity and outer surface and the hydrophilic faces. CDs have long been known to be capable of forming inclusion complexes (Breslow and Overman, 1970; Tabushi, 1982; Saenger, 1984b), which are relatively stable. Since CDs possess negligible toxicity, they have been used as a “host” compound for inclusion of such “guest” compounds as Chlorambucil (Green et al., 1991), Indomethacin (Djedaini et al., 1990) Cholecalciferal (Duchene et al., 1987), Prostaglandin E,

Results The results of elemental analyses of I, II and III are summarized in Table 1. The melting points of I, II and III agreed with reported values (Croft and Bartsch, 1983; Melton and Slesser, 197 1). Compound IV was obtained with a 74% yield of radioactivity, and was immediately used to form V with ANB-

Table 1.

Elementalanalyses of compounds I, II and III Calculated %

Compound iI III

Formula C.,H,OxS C,,H,N,O, CMHu NO,

Found %

C

H

N

S

C

H

N

S

43.33 45.82

5.96 5.90

2.84 -

44.49

6.33

4.21 1.44

45.61 42.97 44.20

5.59 5.74 6.49

4.14 1.69

2.67 -

-

Photoreactive Table Z(a). Biodisttibution

“I In-cyclodextrin inclusion complex

of “‘In-ACAC and “‘In-ACAC-6-amino-6deoxy-a-CD [%ID/g (mean + SD), n = >S per group] I” In-ACAC

Organ

Blood Spleen Liver Kidney Heart Lung Muscle Femur Upper int. Lower int.

4h 3.35 19.74 5.31 3.82 1.16 0.88 0.19 0.92 1.17 1.29

901

(0.47) (1.08) (0.66) (0.33) (0.11) (0.13) (0.02) (0.16) (0.19) (0.20)

“‘In-ACAC-6-amino-ddeoxy-a-CD

24h 0.94 15.26 4.67 2.98 0.37 0.67 0.16 0.66 0.65 0.78

(0.14) (1.02) (0.78) (0.32) (0.04) (0.09) (0.02) (0.08) (0.09) (0.11)

(IV) in non-tumor

4h

48 h 0.06 4.46 3.61 0.46 0.09 0.42 0.02 0.28 0.54 0.58

(0.02) (0.31) (0.45) (0.04) (0.01) (0.06) (0.01) (0.04) (0.08) (0.071

3.63 1.75 2.11 4.78 0.49 0.61 0.25 0.30 0.97 1.02

(0.44) (0.11) (0.03) (0.80) (0.03) (0.07) (0.03) (0.04) (0.14) (0.15)

24h 0.64 0.79 0.76 4.31 0.24 0.14 0.16 0.15 0.47 0.69

rats*

(IV) 48 h

(0.09) (0.05) (0.10) (0.44) (0.03) (0.03) (0.02) (0.03) (0.07) (O.OSj

0.07 0.42 0.30 4.22 0.06 0.08 0.03 0.06 0.23 0.34

(0.02) (0.03) (0.03) (0.43) (0.01) (0.01) (0.0 1) (0.01) (0.051 io.osj

*Lewis rats. Table 2(b). Biodistribution of “‘In-ACAC-a-CD-MAbTP41.2 and “‘In-DTPA-MAbTP41.2 [%ID/g(mean C SD), n = >5 per group] “‘In-ACAC-a-CD-MAbTP41.2 Organ Blood Spleen Liver Kidney Heart Lung Muscle Femur Upper int. Lower int.

4h 3.94 2.73 3.43 5.94 0.50 1.03 0.09 0.19 0.67 0.54

(0.41) (0.22) (0.41) (0.51) (0.01) (0.15) (0.01) (0.02) (0.09) (0.06)

(VI)

24h

48 h

1.01 (0.16) 1.14 (0.12) 1.74(0.25) 5.38 (0.59) 0.33 (0.02) 0.89 (0.11) 0.13 (0.02) 0.21 (0.02) 0.59 (0.07) 0.42 (0.061

0.39 (0.05) 0.74 (0.05) 1.oo(0.22) 4.97 (0.54) 0.29 (0.31) 0.39 (0.05) 0.09 (0.01) 0.20 (0.02) 0.39 (0.05) 0.39 to.051

in non-tumor rats*

“‘In-DTPA-MAbTP41.2 4h 3.64 2.99 5.54 3.51 0.26 0.77 0.12 0.30 0.72 0.71

(0.04) (0.33) (0.68) (0.42) (0.04) (0.09) (0.02) (0.64) (0.09) to.091

24 h

1.78(0.04) 1.96 5.24 3.94 0.20 0.39 0.07 0.29 0.48 0.51

(0.27) (0.66) (0.48) (0.02) (0.06) (0.01) (0.04) (0.06) (0.071

(VII) 48h 0.72 1.25 3.14 3.17 0. I7 0. I2 0.06 0.15 0.42 0.38

(0.09) (0.17) (0.48) (0.46) (0.02) (0.03) (0.01) (0.01) (0.06) (0.05)

*Lewis rats.

et al., 1991) and many other organic compounds in order to increase the stability and solubility of these compounds (Menard et al., 1988). More recently, CDs have been used in column chromatography for separation of optical isomers in analytical chemistry (Valsami et al., 1990; Szejtli, 1988). (Wiese

In vitro stability The in vitro hydrolysis of compounds (VI) and “‘In incorporation into HSA-transferrins at equilibrium times of 4, 8 and 24 h of incubation with HSA at 37°C were found to be 3, 7 and 26%, respectively. It has been reported that “I In-ACAC can serve as a “guest” for a-CD (Wang et al., 1991). The mechanism of this inclusion complex formation is related to the hydrophobicity, relative size and geometry of “‘In-ACAC in relation to the a-CD cavity (5.7A). Arylazides (arylnitrenes) are known photoaffinity labeling reagents (Imai et al., 1990; Lewis et al., 1977; Schrock and Schuster, 1984). The cl-fluoro-3-nitrophenyl azide and 4-azido-2nitrophenol were first used by Knowles and his coworkers for photoaffinity labeling of a specific antibody (Fleet et al., 1969). The ANB-NOS, a new form of arylazide, is chemically stable, not drastically susceptible to photochemical rearrangement and can be photolyzed at wavelengths of 320-350nm, which are less likely to damage the antibody as compared to the alkyl azides, which have absorption maxima of 200-290 nm (Knowles, 1972). Photolysis of the aryl nitrene is smooth, efficient and photolytic generation of the nitrene at the binding site of the antibody leads to specific labeling of the

antibody molecule. The possible mechanisms of the photoreactive labeling arylnitrene with the antibody involve either direct insertion of a C-H bond, or nucleophilic attack by the antibody (Knowles, 1972). The enzyme amylases in salivary glands and the pancreas can hydrolyze 1,4-a-glucoside bonds in starch, glycogen and CDs in vivo to produce maltose, which is then converted to glucose. However, there was a significant difference between the metabolism of starch and CDs. CDs are completely resistant towards r!I-amylase, since they do not contain endgroups susceptible to the attack of this enzyme. a-Alylases, are capable of hydrolyzing the CDs, but usually at a slow rate (Pitha et al., 1983). Following oral administration of [‘4C]starch, [‘4C]a-C0 and [‘4C]j-CD in rats, the [“Clstarch was rapidly hydrolyzed and broken down to [‘4C]glucose, then [‘4C]C02. The hydrolysis of [“C]a-CD as well as [‘4c]/I-CD was, however, slow (Anderson et al., 1963). The same conclusion was reached by Gerloczy et al. (1982) and Szejtli et al. (1980), who fed rats with [*4c]glucose, and [“C]fl-CD and measured the radioactivity level of blood and exheld air. Our studies also revealed that MABTP41.2 conjugated to “‘In-ACAC-CD (V) was relatively hydrolyzed slowly in vitro.

The data reveal that ‘I’ In-ACAC can form a stable inclusion complex with an a-CD derivative (IV), which can then be reacted with the photoreactive compound ANB-AOS. ANB-AOS is readily conjugated with MAbTP41.2 by photoaffinity labeling by U.V. irradiation, instead of the conventional chemical

THEOWRES. T. WANG et al.

902

covalent bond formation method. This new route of radiometallic labeling of MAb can reduce the liver concentration by one-third as compared to the “‘InDTPA method of MAb labeling. This difference in biodistribution between VI and VII is not due to differences in immunoreactivity, as previously biodistribution studies Performed with radiolabeled antimelanoma MAbTP41.2 exhibiting immunoreactivity values varying from 50 to 90% did not result in alteration in either liver or blood concentrations. Based on the stability data which demonstrate relatively low hydrolysis of CD at 8 h, but significant hydrolysis at 24 h, CD may be more useful for labeling antibody fragments rather than whole antibody. These studies suggest that this new technique of radiometallic bifunctional labeling of MAB warrants further investigation.

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