Proc. Nati. Acad. Sci. USA Vol. 77, No. 4, pp. 2219-2223, April 1980 Medical Sciences Macromolecular l-adrenergic antagonists discriminating between receptor and antibody (polymeric drugs/antihormone antibodies/,-adrenergic receptors/steric strain/dextrans) JOSEF PITHA*, JORDAN ZJAWIONY*t, ROBERT J. LEFKOWITZt, AND MARC G. CARONt *National Institute on Aging, National Institutes of Health, Gerontology Research Center-Baltimore City Hospitals, Baltimore, Maryland 21224, tInstitute of Organic Chemistry, Technical University (Politechnika), Zwirki 36, 90-924 Lodz 40, Poland; and tHoward Hughes Medical Institute Laboratories, Department of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Communicated by Curt P. Richter, December 7,1979 ABSTRACT The P-adrenergic antagonist, alprenolol, was Dihydroalprenolol-SA-dextran. A solution of sodium hy- attached in an irreversible manner to macromolecular dextran droxide (2 g) in water (10 ml) was added to the solution of via side arms that differed in length. The ability of these mac- dextran romolecules to bind to the #-adrenergic receptor of frog eryth- (10 g) in water (100 ml). Epichlorohydrin (5.5 g) was rocytes and to catecholamine-binding antibies raised against added and the mixture was stirred at 60'C for 2 hr. After partially purified receptors was studied. Compared to the parent neutralization with saturated aqueous monosodium phosphate, dug the potency of binding of macromolecular alprenolol to 1 M sodium thiosulfate (60 ml) was added and the mixture was the receptor decreased about 1/10, 1/600, and 1/8000 when the stirred overnight at room temperature. After dialysis and length ofthe arm separating alprenolol from the dextran moiety clarification the supernatant was freeze-dried. The resulting was 13, 8, and 4 atoms, respectively. In contrast, the binding potencies of the parent drug and of all its macromolecular de- material (9.96 g) had 1.54% S-i.e., 1 mg contained 0.24 Aumol rivatives for the antibody were within the same order of mag- of thiosulfate residue. Sodium bicarbonate (5 ml, 0.1 M) was nitude. Thus, conversion of a drug to a macromolecular form added to the solution of thiosulfate dextran derivative (1 g, 0.24 may not only sustain its binding activity but may also leal to mmol of thiosulfate residue) in water (10 ml), followed by the a higher selectivity. The macromolecular derivatives described addition of dithiothreitol (462 mg, 3 mmol) in aqueous EDTA here may be suitable probes for investigation of the location and (4 ml, 1 mM). The mixture was stirred for 40 min at room of the molecular properties of the binding sites for P-adrenergic temperature and then dialyzed for 2 hr against nitrogen-purged drugs. water, and the pH of the solution was adjusted to 5 with acetic Conversion of a drug into macromolecular form affects its acid. Alprenolol hydrochloride (257 mg, 0.9 mmol) was then distribution and fate in the organism (1-4). Hydrophilic mac- added and the solution was stirred at 950C for 2 hr. During this romolecules are barred by the lipid bilayer from penetrating time a solution of potassium persulfate (243 mg, 0.9 mmol) in into cells and remain in the extracellular fluid until endocytosed water (1 ml) was added. After cooling, sodium borohydride (35 or excreted. For drugs that act on target tissues by binding to mg, 0.9 mmol) was added and the solution was stirred over- specific receptors located on the cell surface, the exclusion from night. After dialysis and clarification, freeze-drying gave 0.65 the cell interior and longer circulation time may be of distinct g of the material which had maxima in the ultraviolet spectrum pharmacological advantage. In this work we have studied the identical to alprenolol (271 and 278 nm). The material had f3-adrenergic blocking drug alprenolol (Scheme I). This drug 0.51% S-i.e., 1 mg contained 0.16 Amol of S. From absorbancy binds to f3-adrenergic receptors located on the cell surface (5) data it was calculated that 1 mg of material contains 0.21 ,mol and can also bind with similar affinity to catecholamine-binding of alprenolol; by calculation the material should contain 0.29% antibodies that have been raised against partially purified re- N, but 0.73% N was actually found. ceptor preparations (6). Alprenolol was covalently bound to Dihydroalprenolol-MA-dextran. Aqueous sodium hydroxide dextran-and, by varying the length of the linkage arm between (38 ml, 40%) and 2-aminoethyl hydrogen sulfate (40 g) were the drug and soluble carrier (Scheme I; LA, longarm; MA, added to a solution of dextran (5 g) in water (5 ml). The mixture medium arm; SA, short arm), we obtained macromolecules with was placed in a pressure bottle and heated overnight at 900C. different affinities for 13-adrenergic receptor and the cate- After cooling, the mixture was neutralized with diluted hy- cholamine-binding antibodies. The ability of a modified drug drochloric acid and dialyzed at first against aqueous NaCl (10%) to discriminate between related binding sites such as the f3- and then against water. Centrifugation and freeze-drying of adrenergic receptor and these antibodies may find applications the supernatant gave 4.69 g of the material that had 1.59% in analytical measurements or in eventual therapeutic man- N-i.e., 1 mg of material contained 1.1 limol of aminoethyl agement of certain diseases. group. Carbonate buffer (20 ml, 1 M, pH 9.7) was added to the so- MATERIALS AND METHODS, lution of 2-aminoethyldextran (2 g) in water (20 ml). The Materials. Dextran of average Mr 40,000 and racemic al- mixture was cooled to 40C, N-acetyl-DL-homocysteinethio- prenolol hydrochloride were used in all experiments. Unless lactone (1 g) was added, and stirring at 4VC was continued otherwise indicated dialyses were against water at 4VC and in overnight. Dialysis followed by clarification and freeze-drying an exhaustive manner. Clarifications were by centrifugation yielded 1.44 g of product that contained 0.96% S and 0.61% at 10,000 rpm for 10 min. N-i.e., 1 mg of material contained 0.3 Amol of N-acetylho- mocysteine residue and 0.13 ,mol of aminoethyl group. When The publication costs of this article were defrayed in part by page the same synthesis was performed at room temperature, 1.8 g charge payment. This article must therefore be hereby marked "ad- of the material was obtained, containing 1.26% S. The latter vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: LA, long arm; MA, medium arm; SA, short arm. 2219 Downloaded by guest on September 28, 2021 2220 Medical Sciences: Pitha et al. Proc. Natl. Acad. Sci. USA 77 (1980) CHI=CH -CH2 ,+ Alprenolol O-CH2-CHOH-CH2-NH-CH (RI I CH3 CH3-CH2-CH2 -- (RI Dihydroalprenolol D e x t -O-CH2-CHOH-CH2-S-CH2-CH2 CH2- IRI r a Dihydroalprenolol-SA-dextran n CH3 D CO e x NHIH t -O-CH2-CH2-NH-CO-CH-CH2-CH2-S-DC12-CH2 CH2 IRI r a Dihydroalprenolol-MA-dextran D n e x t-O-CH2-CHOH-CH2-O-CH2-O12-CH2-CH2-0-CH2-CHOH-CH2-S-CH2-CH2-CH2--- (RI r a Dihydroalprenolol-LA-dextran n Scheme I product was freshly reduced with dithiothreitol and condensed starting pH was adjusted to 7, the condensation reaction lowered with alprenolol as described for dihydroalprenolol-SA-dextran. it to 6, but amounts of alprenolol incorporated were only half From 0.4 g of material (0.3 mmol of mercapto group), we ob- of the above. tained 0.25 g of the product that had alprenolol-type maxima Binding of [3HJDihydroalprenolol and Macromolecular in ultraviolet spectrum (271 and 278 nm) and had 0.87% N and Alprenolol to Receptors and Antibodies. [3H]Dihydroalpre- 0.64% S. From that data it was calculated that 1 mg of material nolol binding to the various preparations was assessed as de- contained 0.13 /Amol of alprenolol residue, 0.2 ,umol of total S, scribed (6-8) with the following modifications. Purified frog and 0.29 gmol of aminoethyl group. erythrocyte membranes (7) suspended in 25 mM Tris-HCI, pH Dihydroalprenolol-LA-dextran. A solution of sodium hy- 7.4/2 mM were incubated at 25°C for 30 min with in water Mg9l2 droxide (2 g) (10 ml) and 1,4-butanediol diglycidyl [3H]dihydroalprenolol in the presence and absence of various ether (10 ml) were added to a of (10 in water solution dextran g) concentrations of competing ligands. Bound radioactivity was (50 ml). The mixture was stirred for 90 min at room tempera- determined by rapid collection of the incubation mixtures on ture and neutralized with saturated aqueous monosodium glass fiber filters. Binding to the antiserum (no. 349, heat in- phosphate. Sodium thiosulfate (60 ml, 1 M) was then added and activated at 56°C for 30 min) was determined as described the mixture was stirred overnight at room temperature. The above because it was found that the antibodies could be retained resulting alkaline solution (pH 11.5) was again neutralized, on glass fiber filters. Binding of [3H]dihydroalprenolol to sol- dialyzed, clarified, and freeze-dried as above. The product (9.11 ubilized preparations of frog erythrocyte ,3-adrenergic receptor g) contained 1.53% S-i.e., 1 mg of material contained 0.24 in 1% digitonin/100 mM NaCl/10 mM Tris-HCI, pH 7.4 was ilmol of thiosulfate residue. The reduction of the product and assessed at condensation with alprenolol was performed as above. From 4°C by incubating [3H]dihydroalprenolol (5-10 nM) with and without competing of receptor- 1 g (0.24 mmol of thiosulfate residue) of starting material, 0.8 drugs.