Distribution of hexamethonium and other quaternary ammonium compounds in cartilage.

Vol. 176, No. Printed in (I.S.A ASGHAR. KHURSHEED AND LLOYD J. Ro’rH: Distribution of hexamethonium and other (luaternary anhIlloIliufli compounds in (alt ilage. .J. Pliarmacol. Ex1). Ther. 176: 83-92. 1971. N-methyl-C’4and H3-labeled hexamethonium was administered to rats and mice in a dose of 10 to 30 mg/kg. Tissue sections for whole body and cellular autoradiography were cut from animals sacrificed at various intervals after injection. In contrast to the distribution pattern of most drugs hexamethonium was found to he preferentially accumulated in certain avascular cartilaginous tissues, including those of intervertebral discs. epiphyseal and articular cartilages of hip. knee and other joints. costal and art icular cartilage of the ribs, circular cartilage of trachea and nasal and ear cartilages. On the other hand, the compact bone itself or the bone marrow of femur and other hones showed little radiodensity in the autoradiograms. A similar pattern of distribution is observed after C’1-decamethonium. Cellular autoradiograms and hi.stochemical studies showed localization of the labeled hexamethonium in areas rich in acid mucopolvsaccharides. Hexamethonium was also found. in vitro, to bind strongly to chondroitin sulfate. It is hypothesized that the diquaternary ammonium compounds, tinder physiologic conditions, are l)ollfld to the polyanionic mucopolysaccharidc components of cartilaginous tissue. We report here a skeletal distribution pattern for the quaternary ammonium compounds, hexamethonium and decamethonium. In contrast to the distribution pattern of most drugs, hexamethonium preferentially accumulates in certain avascular cartilaginous tissues, whereas relatively little activity is found in blood-rich bone marrow. METHODS. Materials. N-methyl-C” or -H’ labeled hexamethonium and C14-N-methyl-labeled decamethonium were obtained from New England Nuclear Corporation (Boston, Mass.). A radiopurity exceeding 99% was reported by the supplier which was confirmed in this laboratory by (ICscending paper chromatography with the solvent Received for publication July 14. 1970. 1 This investigation was supported by U.S. Public Health Service Grant NB-05668-04. 2 Preliminary reports of these studies were presented at the 1969 Meeting of the American Society of Pharmacology and Experimental Thempeuties (Pharmacologist 11: 293, 1969) and the January, 1970 Meeting of the Western Pharmacology Society at San Diego, Calif. Send reprint requests to: K. Asghar, Ph .1)., I)epartment of Pharmacology, University of Chii’ago. 947 E. 58th St.. Chicago. Ill. 60637. svsteiii of ?l-i)lltaflol saturated with water and HC1 (9: 1). C”-labeled choline (carbon 1 and 2) was obtained from Schwarz BioResearch, Inc. (Orangeburg, NY.). Unlabeled chondroitin sulfate and collagen were obtained from Nutritional Biochemicals Corporation (Cleveland, Ohio). Holtzman white male rats, weighing 140 to 200 g. and white mice, weighing 20 to 25 g (The Jackson Laboratory. Bar Harbor, Maine) (A/J strain), were used. Pregnant mice (inbred DBA/2J strain), weighing 27 to 35 g. were obtained from Dr. William Boggan, Department of Pharmacology, University of Chicago. Preparation of whole body autoradt.ograms. Met hvl-C”-labelcd hexamethonium was administered to rats and mice, (10-20 mg/kg, approximately 2-10 , c). The drug, in saline solution, was injected into the femoral vein of rats anesthetized with 50 ing/kg of j)entoharbital i.p. Hexamethoniun: was administered iv, to conscious mice. Whole body autoradiograms were prepared by the technique of TJllberg (1954, 1958). The rats were sacrificed, at various intervals after drug administration, by immersion into liquid nitrogen. The frozen rats were transferred to a cold room maintamed at -8 to -10#{176}C. For mounting the carcass, a s ecially designed brass stage was cooled on Dry Ice. A thin layer of rayon-cellulose gauze soaked 84 ASGHAR AND ROTH Vol. 176 in vater was spread out on this stage. The frozen animal was placed on the gauze in the desired 1)osition and surrounded by cooled carboxymethylcellulose (CMC) gel and placed in contact with Dry Ice for a few hours until the CMC hardened. after which the mount was trimmed to a cuboidal block. The stage with the CMC-encased rat was figed in position on a heavy microtome (type: Jung. A. G.. Heidelberg, Germany). Fortyto 6O-/L thick sections were then (‘Ut by a fine microtome blade, picked up on the adhesive tape and left in the cold room to air dry overnight. They were then apposed to no-screen Kodak X-ray film ( N-54) for exposure. A steek of 10 section sets, ‘ith a thick 1)iece of filter paper between each set, v:ts compressed between two brass plates with bin(Ier clips (I1)T. Manufacturing Corporation, Carlstadt, N.J.) and stored in dark in the COl(l 100111 for an al)Propriate interval. usually 1 to 6 weeks. The sections and the photographic emulsions removed from the cold room. and the separated X-ray film :is processed. Preparation of cellular a utoradiogra ins. Nmet hvl-H’-labeled hexamethionium was administered i.p. to mice in a (lose of 30 mg/kg (2.5 c/g). The dry mounting autoradiography procedure and its usefulness for the stu(ly of diffusible compounds hits been (liscusse(l recently (Roth an(l Stumpf. 1969). Two-micron thick sections were cut, freeze dried tnt! dry mounted in an atmosphere of low relative humidity onto dried photographic emulsion-(’oat4#{176}d slides (Stuinpf and Roth. 1967, 1968). After photographic exposure for 2 to 3 weeks at -15#{176}C the autoradiograms were developed, and issues were staine(I for acid mucopolysaccharides and collagen. The following staining procedures (Wagner and Smith, 1967; J una, 1968) were used: hematoxvlin and eosin, Alcian Blue. Alcian Blue. Periodic acid-Schiff reagent, aldehvde fuchsin Alnan Blue, perio(li(’ acid-.Schiff (PAS) reagent and Van GiesOn’s stain. Binding of hexamethonium to c/wndroitin. The binding of C#{176}4-labeled hexamethonium to chon(lromtin sulfate was studied by dynamic dialysis and equilibrum dialysis. The dynamic dialysis method used was basically that of Meyer and Cuttman (1968. 1970a,b). Cellulose dialysis bags (Oxford Laboratories, San Mateo, Calif.), with a o e diameter 4.8 m , were used, Twenty microliters containing 58 g of hexamethonium (5.9 X 10#{176} cpm) were added to 2 ml of 0.04 M phosphate buffer solution either in the absence or lresence of chondroitin sulfate at 25-26#{176}C. This solution Was transferred to a dialysis sac which was suspended in an Oxford dialyzer containing 300 or 600 ml of buffer medium containing an additional 0.10 M sodium t’hlonde. The external medium was wholly replaced evei’y 40 minutes thereby maintaming almost sink conditions. Equilibrium dialysis (Goldstein et (Ii., 1968) ViLS conducted with dialysis sacs as above. One milliliter of 1.0% chondroitin sulfate solution was dialyzed against 500 ml of external fluid containing 1.0 g/ml to 0.1 mg/mI of hexamethonium dichiori(le. Preliminai’v experiments had indicated that equihil)rium had been i’eached at 24 hours, and samples were withdrawn at this time from inside 111(1 outside the dialysis sac and analyzed by liquid scintillation counting. A negligible fraction of the total hexamethonium \ 5 l)OIlfl(I to the dialysis sill, itself. Radiochemical (1?lalysi . We have measured the extent of drug accumulation in the cartilage by (letermining the ratio of the concentration of the drug in the epiphyses and in the diaphyses. The el)iphyses are not wholly cartilaginous and contain son e ossified tissue. The actual cartilage levels of the drug, therefore. would be much higher than the concentration of the drug in epiphysis. N-nwthvl-C”-labeled hexamethonium (20 mgI kg) a(lnhinistered i.p. to mice. At various intervals the mice were killed by freezing in liquid nitrogen, and the femurs were excised. The bone (11(1 cart ilage samples were freed of adjoining soft tissue and dried at 80-100#{176}C to a constant vight. After drying. the distal condyle segment. at )l )roxi matehy 1.5 mm in length, containing mainly the epiphvsis, was cut out. A sample of diaphysis along with the bone marrow was obtained from the (‘enter of the femur shaft. These samples were weighed separately, pulverized in a glass homogenizer under similar conditions to a fine powder in 0.01 N hydrochloric acid-90% ethanol and centrifuged. This procedure was found to remove the labeled hexamethonium completely from the mouse bone/cartilage. A 0.5-ml aliquot of the supemnatant was suspended in Cab-O-Sil toluene phospor solution and counted for 10 minutes in a Packard Tri-Carb spectrometer at the instrumental settings of 0501000, 12%. The toluene phosphor solution had the following composition: 2,5 diphenyloxazole, 4g; 1 ,4-bis[2-(5-phenyloxazolyl)]benzene, 400 mg; in 1 liter of toluene. The counting efficiency was deterinined by internal standardization and found