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Preparation of Sulfhydrylborane-Dextran Conjugates for Boron Neutron Capture Therapy Anders Holmberg'lt and Lennart Meurlingt Kabi Pharmacia Diagnostics AB, S-751 82 Uppsala, Sweden, and Mercodia AB, 5-752 28 Uppsala, Sweden. Received April 12, 1993" ~~~
_ _ _ _ ~
This study presents a carrier system for boron, potentially useful in boron neutron capture therapy (BNCT). NazBlzHllSH (BSH) was covalently coupled to dextran derivatives. This was accomplished in two ways. The first method comprises activation of dextran with l-cyano-4-(dimethylamino)pyridine (CDAP) with subsequent coupling of 2-aminoethyl pyridyl disulfide (method A). The thiolated dextran could then couple BSH in a disulfide exchange reaction. In the second procedure, dextran was derivatized to a multiallyl derivative (method B) which reacted with BSH in a free-radical-initiated addition reaction. The assessment of boron content of the conjugates was done by elemental analysis of sulfur and atomic spectroscopy of boron (ICP-AES). With method A, only limited numbers of boron cages could be coupled (10-20 cages per dextran chain). With method B, 100-125 boron cages per dextran chain was obtained, corresponding to 1200-1500 boron atoms per dextran chain. This result makes this derivative a promising template for use in the development of BNCT agents.
INTRODUCTION The principle for boron neutron capture therapy (BNCT) for treatment of cancer is the nuclear reaction that occurs when 1OB is irradiated with thermal neutrons. This reaction yields an unstable intermediate, llB, which undergoes fission, yielding 'Li and 4He. One of the conditions for this therapy to be successful depends on the ability to transport sufficient concentrations of boron to the tumor tissue and preferably localize the boron into the cell. The induced radiation should then ultimately kill the tumor. The effects and medical possibilities of neutrons were described by Locher (Locher, 1936) and further by Bale (Bale, 1952). Clinical trials were first performed in the United States during the period 19511961, however with discouraging results. In the last two decades treatment of brain tumors using BNCT has been performed in Japan (Hatanaka, 1990) using improved boron compounds and neutron irradiation techniques. Promising results have been obtained and the interest for BNCT is increasing. One of the main problems in BNCT is the delivery of boron to the target tissue. Thus many workers are involved in the construction of carrier systems with capacity for enhanced loads of boron. A variety of compounds have been developed as boron carriers, for example antibodies (Bale and Spar, 1967; Hawthorne et al., 1976),amino acids (Ichihashi et al., 1982), porphyrins (Kahl et al., 19901,and low-density lipoproteins (Laster et al., 1991). In the present study we have investigated the possibility to use dextran and BSH as a template for a BNCT agent. BSH is currently the best documented boron compound for BNCT, and dextran has previously been used in numerous studies as a microcarrier for both low and high molecular weight substances (De Belder, 1990;
* The author to whom correspondence should be addressed. Telephone: 4618163587. Fax: 4618126419. + Kabi Pharmacia Diagnostics AB. t Mercodia AB. * Abstract published in Advance ACS Abstracts, October 1, 1993. 1043-1802/93/2904-0570$04.00/0
Foster, 1975; Hirai et al., 1989; Jost and Yaron, 1974; Melton et al., 1987)including tumor targeting applications (Bernstein et al., 1978; Hurwitz et al., 1980). Antibodies have been boronated via a dextran bridge (Pettersson et al., 1989). The activation methods employed have usually been periodate oxidation and conventional cyanogen bromide activation. Dextran is also well-known as a blood plasma volume expander. The preparation of BSHdextran conjugates was done with two different methods, dextran activation with CDAP (Kohn and Wilchek, 1983) and subsequent thiolation with 2-aminoethyl pyridyl disulfide (method A) and reaction of BSH with allyl dextran (method B). The mechanism for the addition of mercaptans to double bonds in the presence of free-radical initiators has been described earlier (Stacey and Harris, 1963). The analysis of the boron content was made by elemental analysis of sulfur (Holmberg et al., 1992) and atomic spectroscopy of boron, ICP-AES, (Moore, 1990). With method A, only moderate numbers of boron cages could be coupled, that is 10-20 boron cages per dextran chain. However with method B, 100-125 boron cages per dextran chain were coupled with retained solubility of the conjugate. This corresponds to 1200-1500 boron atoms per dextran chain. The high carrying capacity of this derivative makes it potentially useful as a BNCT agent. EXPERIMENTAL PROCEDURES
Materials. Dextran T70 (Pharmacia Biotech AB, Sweden) with an average molecular weight of 70 kDa was salt (CDAP) used. l-cyano-4-(dimethylamino)pyridinium for dextran activation and 2-aminoethyl pyridyl disulfide (AE-PDS) for thiolation were gifts from Kabi Pharmacia Diagnostics AB, Sweden. Triethylamine (TEA) from Rietel De Haen AG, Germany, was used. Na2B12H11SH (BSH) from Boron Biologicals was used as the boron compound. The separation and purification were carried out with gel filtration on NAP-5 G-25 and PD-10 G-25 disposable Sephadex G-25 columns from Pharmacia Biotech AB Sweden. The purity of the products was analyzed on a Sephadex G-25 1X lOcm column, Pharmacia Biotech AB, connected to a Shimadzu refractive index detector. 0 1993 American Chemical Society
Bioconjugate Chem., Vol. 4, No. 6, 1993 571
Technical Notes
UV absorbance measurements were done with a Beckman UV spectrophotometer. ICP-AES was done with a Varian ICP-AES Liberty 200 instrument (Analytica AB, Sweden). Methods. CDAP Method (method A ) . Dextran T70 (250 mg, 3.5 pmol) was dissolved in 25 mL of distilled water and mixed with 125 mg (0.55 mmol) of CDAP for 10 s. Triethylamine (75pL, 0.5 mmol) was added dropwise and the reaction mixture was stirred for 150 s a t room temperature. A 250-pL sample of the reaction mixture was withdrawn and immediately mixed with the coupling solution containing 2.5 mg of AE-PDS in 250pL of 0.4 M Na2HP04, pH 8.0. The conjugation was performed a t room temperature for 120 min. After the conjugation low molecular weight residues were removed on a NAP-5 G-25 column equilibrated with 0.5 M NaC1. The sample was eluted with 0.2 M NaZHP04, pH 8.0. A 75-pL sample of the thiolated dextran was then withdrawn and diluted to 3 mL in 0.2 M Na2HP04,pH 8.0. Dithiothreitol (30 mg) was added and after incubation for 10 min the absorbance was recorded at 343 nm in a spectrophotometer. From the released thiopyridone (E 8080) the subtitution of AEPDS could be calculated. The thiol dextran mixture was then added to a coupling solution containing 20 mg of BSH (0.14 mmol) in 0.5 mL of 0.2 M Na2HP04,pH 8.0, and stirred a t room temperature overnight. After the incubation a new 75-pL sample was taken and diluted to 3 mL in the same buffer as above. The released thiopyridone was measured a t 343 nm and the efficiency of the reaction could be calculated. Finally, the conjugate was purified on a PD-10 G-25 column (equilibrated with 0.5 M NaC1) and eluted with distilled water. The purity of the product was confirmed by analytical chromatography on a 1 X 10 cm G-25 column. The eluate was analyzed with a refractive index detector and was found to be homogenous. Finally the product was lyophilized. The sulfur content was determined by elemental analysis (Kirsten and Nordenman, 1987). From the results obtained, the degree of substitution was calculated and hence the boron content, assuming two sulfur atoms per boron cage. Allyl Bromide Method (Method B ) . Dextran T70 (20 g) was dissolved in 150 mL of distilled water together with 5 g of NaOH and 0.2 g of sodium borohydride. Allyl bromide (35 g, 0.30 mol) was added a t 40°C. The mixture was stirred a t 60 "C for 3 h and then neutralized with acetic acid. The product was purified by repeated precipitations in ethanol and dried to a constant weight under vacuum. The allyl content was determined by bromination of the dextran derivative in water, followed by potentiometric titration of released bromide with silver nitrate. To a solution of 20 mg of allyl dextran in 0.2 mL of distilled water was added 30 mg of BSH (0.13 mmol) and 20 mg of ammonium persulfate. The solution was stirred for 2 h a t 50°C. The reaction mixture was purified by gel filtration on a PD-10 G-25 column according to the manufacturers recommendations. The purity of the product was confirmed by analytical gel chromatography on a 1 X 10 cm G-25 column. The eluate was analysed with a refractive index monitor and was found to be homogenous. Finally the product was lyophilized. The product was analysed for boron content by means of inductively coupled plasma atomic emission spectroscopy (ICP-AES). The sulfur content was determined by elemental analysis.
Scheme I
6c: -
H3C,N.CH3
TEA
Dx-OH
+
+
Dx-0-CIN
111
N
Dx-0-CIN
+
NH,-CHz-CHz-S-S
-Q
-t
IH
Dx-0-C-NH-CH,-CH,-S-S !H Dx-0-C-NH-CH,-CH,-S-S-R
+
O S
Scheme I1
FLaoH
Dx-OH
+
-
CH,=CH-CH,Br
Dx-0-CH,-CH:CH,
BSH
Dx-O-CH,-CH,-CH,-S-R
R = Boron cage Dx = Dexhan
RESULTS The overall reaction of CDAP with dextran and coupling of BSH is shown in Scheme I. With the CDAP method 10-20 boron cages per dextran chain was coupled. This is in agreement with the number of thiol groups introduced (12-20 AE-PDS per dextran chain) and the released thiopyridone after the BSH coupling. The reaction was quantitative. The elemental analysis gave figures from 0.5% to 0.9% sulfur corresponding to a boron content from 10 to 18 pglmg of conjugate (assuming two sulfur atoms per boron cage), that is 10-20 boron cages. Attempts to raise the substitution level by increasing the activation invariably resulted in precipitation either during the activation or in the coupling of AE-PDS. The pH optimum for the coupling of AE-PDS was approximately pH 8. When the activation and the subsequent coupling of AE-PDS could be done without precipitation, the complex remained soluble during and after the disulfide exchange reaction with BSH. In order to increase the amount of boron bound we introduced the allyl bromide method, which is shown in Scheme 11. With method B, the substitution of boron was higher. The ICP-AES analysis gave 150 pg of boron/mg of conjugate. The sulfur content was 3.5-4%. This corresponds to a substitution of 1 W 1 2 5 boron cages per dextran chain or 1200-1500 boron atoms per dextran chain. Since the allyl content was determined to be 3.75 mmollg of dextran derivative (70% of the glucose units had been substituted with an allyl group), approximately 50% of the allyl groups had been substituted with a boron cage. The complex remained soluble in all steps of the synthesis. DISCUSSION In this study we described two methods to couple boron cages to dextran. Method A is easy to perform and very
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fast but has some disadvantages: first, low capacity and, second, the product sometimes precipitates. The precipitation occurring was probably due to cross-linking of the dextran. In the activation step the reactive species reacts further with adjacent hydroxyl groups, thus crosslinking the dextran. This side reaction increases with increasing degree of activation. In the coupling of the thiol compound the precipitation could be explained by S-S formation between dextran chains. The results indicate that the CDAP method as an activator of dextran does not provide enough carrying capacity for boron as required for BNCT. However, the CDAP method can be useful for the preparation of other targeting compounds using dextran and with lower requirements for carrying capacity. The CDAP method gives the same products as the cyanogen bromide reaction. It should always be preferred before the conventional toxic and inefficient cyanogen bromide method. The B method proved to be very efficient as an activator of dextran, giving capacity to couple a high number of boron cages. The general carrying requirement for a BNCT compound (i.e. 1000 boron atoms/conjugate) was exceeded. We believe that the carrying capacity of this derivative can be even more improved by further optimization. The charge of the derivative is anionic, which can be of importance for in vivo localization (Maeda et al., 1992). It is possible to couple a targeting molecule to the boron-dextran, making it tumor-specific, for example epidermalgrowth factor (Andersson et al., 1991)or another tumor-specific molecule. BSH-dextran can thus be seen as a template for a potentially useful BNCT agent. The BSH-dextran derivative might by itself have a potential as a “passiven targeting BNCT agent. The concept of “passive” targeting has been extensively studied and described by Maeda (Maeda et al., 1992) and Seymour (Seymour, 1992). It is based on the increased permeability for macromolecules of tumor vasculature and the impaired or absent lymph drainage. This leads to prolonged retention of macromolecules in tumor tissue (the enhanced permeability and retention effect, EPR). EPR is seen in most solid tumors, and it is probable that EPR is of dominating importance for any tumor targeting compound, specific or passive. The most positive clinical results obtained with BNCT has been with BSH “passively” administered to the tumor even though the mechanism for the affinity of BSH for tumors remains still unclear. The dimer of BSH has been shown to have even stronger tumor uptake than the monomer (Joel et al., 1990). The “poly BSH” derivative described here might show a similar affinity for tumor tissue. Another direct application of boron-dextran could be BNCT of arterio venous malformations (AVM). This treatment possibility has been proposed by Larsson (Larsson, 1988). Lately BNCT for AVM has been performed in Japan by Hatanaka (Hatanaka et al., 1992). The BSH-dextran derivative described here could be very suitable for this purpose. Experiments to evaluate the clinical relevance of the above mentioned boron dextran are in progress and will be reported elsewhere. ACKNOWLEDGMENT This work was supported by Kabi Pharmacia Diagnostics AB, S-75182 Uppsala, Sweden. The author wants to thank Dr. Holmlund a t MERCODIA AB for fruitful discussions and Dr. Weinreich a t IMR, University of Zurich, for supporting the boron analysis.
Holmberg and Meurling
LITERATURE CITED Andersson, A., Holmberg, A., Carlsson, J., Carlsson, J., Ponten, J., and Westermark, B. (1991)Binding of epidermal growth factor-dextran conjugates to cultured glioma cells. Znt. J. Cancer 47,439-444.
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De Belder, A. N. (1990)Dextran, Pharmacia Leo Therapeutics, Uppsala, Sweden.Bernstein,A., Hurwitz,E., Maron,R., Amon, R., Sela,M., and Wilchek,M. (1978)Higher antitumor efficacy of daunomycin when linked to dextran: In vivo and in vitro studies. J. Natl. Cancer Inst. 60, 379-384. Foster, R. L. (1975)Preparation and properties of a soluble trypsin-dextran conjugate. Experentia. 31, 772-773. Hatanaka, H. (1990)Clinical results of boron neutron capture therapy. In Neutron Beam Design, Development and Performance for Neutron Capture Therapy (0.K. Harling, J. A. Bernard, Jr., and R. G.Zamenhof, Eds.) pp 15-21, Plenum Press, New York. Hatanaka, H., Nakagawa, Y., and Sakaki, S. (1992)Endovascular boron-neutron capture radiotherapy for giant arteriovenous malformations. 5th Int. Symp. Neutron Capture Ther. 122, 62.
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Ichihashi, M., Nakanishi, T., and Mishima, Y. (1982)Specific in thermal neutron Killing effectof 10B1-Para-borophenylalanin capture therapy of malignant melanoma: In vitro radiobiological evaluation. J. Invest. Dermatol. 78, 215-218. Joel, D.D.,Fairchild, R. G., Laissue, J. A., Saraf, S. K., KalefEzra, J. A,, and Slatkin, D. N. (1990)Boron neutron capture therapy of intracerebral rat gliosarcomas. Proc. Natl. Sci. U.S.A. 87,9808-9812. Jost, R., and Yaron, A. (1974)Preparation of solubleand insoluble polysaccharide conjugates of peptides with a free alpha amino group. Eur. J. Biochem. 48, 119-129. Kahl, S. B., Joel, D. D., Nawrocky, M. M., Micca, P. L., Tran, K. P., Finkel, G. C., and Slatkin, N. L. (1990)Uptake of a nido-carboranylporphyrinby human glioma xenografts in athymic nude mice and by syngenic ovarian carcinomas in i”unocompetentmice.Proc. 7269.
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Kirsten, W., and Nordenmark, B. (1987)Rapid, automatic, highprecision method for micro, ultramicro, and trace determinations of sulphur. Anal. Chim. Acta 196, 59-68. Kohn, J., and Wilchek, M. (1983) 1-Cyano-4-dimethylamino pyridinium tetrafluoroborate as a cyanylating agent for the covalent attachment of ligand to polysaccharide resins. FEBS Lett. 154, 209-210.
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Technlcal Notes
Laster, B. H., Popenoe, E. A., Pate, D. W., and Fairchild, R. G. (1991)Biological efficacy of boronated low density lipoprotein for boron neutron capture therapy as measured in cell culture. Cancer Res. 51, 4588-4593.
Locher, G. L. (1936)Biological effectsand therapeuticpossibilities of neutrons. Am. J . Roentgenol. Radium Ther. 36, 1-13. Maeda, H., Seymour, L. W., and Miyamoto, Y. (1992)Conjugates of anticancer agents and polymers: Advantages of macromolecular therapeutics in vivo. Bioconjugate Chem. 3,351-362. Melton, R.G., Wiblin, C. N., Foster, R. L., and Sherwood, R. F. (1987)Covalent linkage of carboxypeptidase G2 to soluble dextrans. Biochem. Pharmacol. 36, 105-112.
Moore, D. E. (1990)A review of techniques for the analysis of boron in the development of neutron capture therapy agents. J . Pharm. Biomed. Anal.8, 547-553. Pettersson, M. L., Courel, M. N., Girard, N., Gabel, D., and Delpech,B. (1989)In vitro immunologicalactivity of adextranboronated monoclonal antibody. Strahlenther. Onkol. 165, 151-152.
Seymour, L. W. (1992) Passive tumour targeting of soluble macromolecules and drug conjugates. Crit. Rev. Ther. Drug Carrier Sys. 9,135-187. Stacey, F. W.,and Harris, J. F., Jr. (1963)In Organic Reactions, Vol 150.
