The Pharmacological Studies on Dimetacrine I. Studies on the Absorption, Distribution and Excretion of H3-Labeled Dimetacrine in the Rat

THE PHARMACOLOGICAL STUDIES ON DIMETACRINE I. STUDIES ON THE ABSORPTION, DISTRIBUTION AND EXCRETION OF H3-LABELED DIMETACRINE IN THE RAT

RYOICHI ISHITANI*, KENTARO SAITO* AND HARUO KITAGAWA** *Departmentof Researchand Development, HitachiChemical Company Limited, Honkomagome, Bunkyo-ku, Tokyo **Departmentof BiochemicalPharmacology , Facultyof PharmaceuticalSciences, University of Chiba,Chiba Receivedfor publication March 4, 1970

Dimetacrine, 10-[3(dimethylamino)propyl]-9,9-dimethylacridan, is a tricyclic com pound with a central hexagonal ring instead of the heptagonal ring common to most anti depressants. The synthesis and chemistry of this compound have been reported by Molnar et al. (1).

The unique pharmacological properties of dimetacrine as a reserpine antagonism, central parasympathicolytic action and the occurrence of behavioral changes in cats and rats were observed by Jahn (2). The clinical properties and the value of this drug in the treatment of depressive states, and in a variety of psychic disorders have been well established (3-5). Schatz et al. (6) demonstrated the metabolic fate of dimetacrine by means of colori metric assay based on the method of Herrmann et al. (7). However, the sensitivity of this method is relatively low for detection of dimetacrine and its metabolites, therefore, the present paper was undertaken to determine the metabolic patterns of this drug using radio metric assay. MATERIALSAND METHODS 1. H'-Dimetacrine Labeled dimetacrine with a specific activity of 22 pCi/mg was synthesized according

石谷良一・斉藤堅太郎・北川晴雄 to the method of Eidinoff et al. (8), using a tritium water as labeled precursor. The final product was chemically and radiochemically pure which was determined by mixed melt ing points with authentic dimetacrine and by paper chromatography in the following solvent system-toluene : ether: formic acid (10: 10: 5) and MeOH : 28-29% ammonia water (1: 1). 2. Animals Male Wistar rats (200-250 g) were given 20 mg/kg , (0.44 mCi/kg) of H3-dimetacrine orally or intraperitoneally. All animals used here were fasted overnight before experiments. 3. Assay proceduresof H'-dimetacrinefrom biological materials 1) Drug concentrationsin blood Blood specimens were withdrawn from tail vein at points indicated in Fig. 1. An aliquot of blood (0.02 ml) was adsorbed on filter paper and kept overnight at room tem perature to dryness and then combusted using the combustion apparatus described' else where in this paper. 2) Distribution of the drug in tissues The animals were decapitated at appropriate time intervals after drug administration, the chest was opened and bled as much as possible from the heart. Tissues were removed, blotted, weighed and homogenized in 4 volumes of water with a motor driven Potter Elvehjem homogenizer. An aliquot of tissue homogenates was lyophilized. The materials were weighed, carefully ground, and an aliquot of this materials no more than 50 mg was combusted using a combustion apparatus. The combustion method have been reported by several investigators (9-12) and in this paper, the dry combustion method as described by Baba et al. (13) was used. The resulting tritium water after combustion was collected by condensation in dry ice-acetone mixture and the condensed tritium water was eluted three times into counting vials with dioxane phosphor (14) in the volumes of 10, 5 and 3 ml. Over 98% of the radioactivity, when H'-dimetacrine was added to control tissues prior to homogenization under the condition described above, was recovered from the homogenates. 3) Excretion of drug in urine,feces and bile Urine and feces were collected separately in metabolism cages, in which rats were given H'-dimetacrine in a dose of 20 mg/kg (0.44 mCi/kg) orally or intraperitoneally. For determination of H'-radioactivity in bile, a 24 hour bile was collected from the rats given 20 mg/kg (0.44 mCi/kg) of H'-dimetacrine orally. Radioactivities of feces were measured as the dry combustion method described elsewhere in this paper. Urine and bile were diluted to 50-100 ml with water and radioactivities in aliquots (0.5 ml or 1.0 ml) of diluted samples were measured in counting vials containing 15 ml of dioxane phosphor. Counting H3-radioactivity was counted in the Beckman liquid scintillation spectrometer DPM 100, using a dioxane phosphor. The counts (cpm) were converted to disintegrations per minute (dpm) by the use of the channel ratio method. Using the combustion method, H3-radioactivity was assayed at 45% efficiency and in biliary excretion studies, counting efficiency was 33%.

RESULTS

1. Absorption of H3-dimetacrineand its average half-life in blood Blood specimens of rats given H'-dimetacrine, either orally or intraperitoneally, were withdrawn from tail vein at appropriate times and were assayed for total radioactivity. Appreciable amounts of radioactivity were found in the whole blood 0.5 hour after administration by either routes and the highest concentration was observed at 1 hour after intraperitoneal administration and thereafter declined, with an average half-life of approxi mately 10 hours. On the other hand, the maximum concentration of the drug was found within 1 hour after oral administration and this level was thereafter maintained for additional 6-8 hours and then declined, with an average half-life of approximately 24 hours. In both routes, the levels of the drug declined to about 17-14% of the maximum con centration within 72 hours after drug administration.

FIG. 1. Concentration of total radioactivity in whole blood. Rats were given 20 mg/kg (0.44 mCi/kg) of H3-dimetacrine in the two routes used. 2. Distribution of H3-dimetacrinein tissues After oral administration of H3-dimetacrine, the animals were decapitated at various times indicated and the total radioactivities of tissues were assayed (Tables 1 and 2). There were appreciable concentrations of radioactivity in all tissues at 0.5 hour after administration. The highest concentrations of radioactivity was found at 1 hour after administration but at 3 hours in brain, heart, lung, liver, spleen, kidney, skeletal muscle and adipose tissue of testes. At all time intervals investigated, the concentration of radioactivity (percentage of dose per gram tissue) in the lung was the highest among all tissues investigated. As shown in Table 2, the concentration of total radioactivity in stomach and small intestine was the highest at 1 hour, and 72 hours later, the total radioactivity in these tis sues declined to as little as 1.03% of the dose administered. Recovery of H'-radioactivity from tissues, urine and feces at 1 hour, 3 hours, 12 hours, 24 hours and 48 hours was 63.83%, 56.99%, 53.80%, 52.19% and 71.44%, respectively. In Table 2, the values of blood, skeletal muscle and adipose tissue are not recorded.

TALE 1. Total radioactivity in rat tissues at various times after oral administration of H3 dimetacrine. Fifteen rats were given 20 mg/kg (0.44 mCi/kg) of H3-dimetacrine orally. At inter vals three rats were killed for assay. Results are expressed as percentage of dose per gram of tissue. Average values are given with the range shown in brackets.

3. Excretion of radioactivityafter H'-dimetacrine administration Rats were given H'-dimetacrine orally and intraperitoneally. Urine and feces were collected at 12, 24 and 48 hours after administration and assayed for their total radio TABLE2. Total radioactivity in rat tissues, small intestine and excreta at various times after oral administration of H3-dimetacrine. Fifteen rats were given 20 mg/kg (0.44 mCi/kg) of H3-dimetacrine orally. At intervals three rats were killed for assay. Results are expressed as percentage of dose per total weight of specimens. Average values are given with the range shown in brackets.

TABLE3. Urinary and fecal excretion of radioactivity after intraperitoneal administration of H3-dimetacrine. Three rats were given 20 mg/kg (0.44 mCi/kg) of H3-dimetacrine intraperitoneally. Results are expressed as percentage of dose. Average values are given with the range shown in brackets.

activity (Tables 2 and 3). When administered by the two routes, approximately 70% of the doses was excreted in urine and feces within 2 days after treatment. As shown in Tables 2 and 3, feces at 1, 3 and 12 hours and urine at 1 and 3 hours later were difficult to collect, therefore, radioactivities of these samples were not determined. Within 24 hours after oral administration, 24.15% of the dose was eliminated in bile. The decrease of the radioactivity in the liver TABLE4. Biliary excretion of radioactivity during this period probably reflects excre after oral administration of H3-dimeta crine. tion into the bile. Rats were given 20 mg/kg (0.44 mCi/ kg) of H3-dimetacrine orally. Bile was DISCUSSION collected for 24 hours and assayed for total radioactivity. Results are ex The highest concentration of radioac pressed as percentage of dose. tivity in whole blood after oral administra tion of H'-dimetacrine was observed at 1 hour and continued for 6-8 hours there after. The prolonged action of the pharma cological effects of dimetacrine perhaps is the reflection of its long half-life in the blood. At 3 hours after oral administration, the total radioactivity in the liver was 11 9% of the administered dose, and declined to 1.09% within 48 hours; on the other hand, 24.15% of dose was excreted in bile within 24 hours. These results suggest that the portal circula tion is involved in absorption and excretion of this drug. In studying the distribution of this drug, the sum of radioactivities of brain, heart, lung, liver, kidney and spleen was 1.54% of the administered dose at 48 hours after oral ad ministration; accumulation of the drug was not observed. The total radioactivities in excreta were found at the levels of 68.87 and 75.04% of dose at 48 hours after administra tion by the two routes, i.e., i.p. and p.o. Therefore, these results suggested that H'-dimeta crine was absorbed and excreted rapidly when the drug was administered. Total recovery of H'-radioactivity was 71.44% of dose at 48 hours after oral adminis tration and the remainder probably was located in the large intestine. In the present studies, the quantity of H'-dimetacrine found in excreta was large com pared to that found by others (6). A few reports are available on the pharmacological studies and metabolism of imi pramine which possesses similar chemical structure to that of dimetacrine. Herrman et al. showed that imipramine was slightly excreted in urine after oral administration in clinical use; however, another experiment (15) using radiometric assay demonstrated that 40% of dose was excreted at 24 hours after administration and 70% at 72 hours later in urine of patients treated with C14-imipramine. In general, the radiometric assay used here is superior to the chemical method reported by Schatz et al. because of its sensitivity.

SUMMARY 1. The total radioactivity of whole blood in rats given H3-dimetacrine orally reached to a maximum level within 1 hour and remained so for 6-8 hours thereafter; in addition, the average half-life was 24 hours. By the intraperitoneal administration, the maximum concentration was seen at 1 hour and thereafter declined with an average half-life of ap proximately 10 hours. 2. The distribution of H3-dimetacrine in tissues was studied after oral administration. The highest H'-radioactivity in most specimens with the exceptions of stomach, blood and small intestine was observed at 3 hours later and total radioactivity of lung (as % dose /g tissue) was the highest at appropriate times investigated. The concentration of total radioactivities in stomach and small intestine were 1.03% at 48 hours after administration. H3-recovery from all specimens, excluding the values of blood, skeletal muscle and fat, was 71.44% at 48 hours after administration and excreta were accounted for 68.87% of total recovery. 3. After intraperitoneal injection, 40.52% and 75.04% of dose was excreted in ex creta within 24 hours and 48 hours, respectively. 4. Biliary excretion was 24.15% within 24 hours after oral administration. Acknowledgements:The authors wish to acknowledge the advice and encouragement of Prof. S. Yama gishi and Dr. Y. Koyama (Department of Microbiologyand Chemistry, Faculty of Pharmaceutical Sciences, University of Chiba).

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