Time course observation on metaproterenol distribution in rats.

The plasma 3H-radioactivity level showed a high value 1 hr after intra peritoneal injection of 3H-metaproterenol nd it l wered lineraly to reach 40% of the peak level 5 hr after administration. Radioactivity distributed to the kidneys and liver was markedly higher than that in other organs. Forty per cent of the amount given orally was excreted in the 24 hr urine specimen. The major part of metapro terenol was excreted in urine as an unchanged compound in the early stages after administration and the remainder in the form of glucuronide in the late stages. It is concluded that the absorbed metaproterenol remains unmetabolized for a consider able length of time in the body and high plasma levels are maintained for a relatively long period. Metaproterenol [1-(3, 5-dihydroxyphenyl)-1-hydroxy-2-isopropyl aminoethane], a sym pathomimetic drug has been utilized in clinical practice as a bronchodilator. Based on clinical experience, metaproterenol shows a long-lasting response against asthmatic attacks as compared with other sympathomimetics (1-4). Dengler reported that metaproterenol was excreted in the urine of rats partly in the form of glucuronide (5), however, there is a paucity in information regarding the kinetics of metaproterenol. For this reason, the present study was undertaken in order to elucidate the time course of the 3H-metaproterenol distribution pattern in rats following intraperi toneal and oral administrations. Urinary excretion of 3H-radioactivity and its metabolites after administration of the agent were also investigated. MATERIALS AND METHODS According to the Wilzbach method (6), 500 mg of metaproterenol was exposed to 20 Ci of tritium gas for 5 days. The compound was purified further by removal of labile tritium. 3H-metaproterenol was identified by radiothinlayer chromatography on silica gel in two solvent systems; [ethylacetatem-ethanol-ammonia water (17: 6: 5), ethanol ammonia water-water (25:4: 3)]. Wistar rats of both sexes weighing from 200 to 300 g were used. 3H-metaproterenol (sp. act. 14 pc/mg) was administered in a dose of 50 mg/kg i.p. The drug (sp. act. 16 ,ug/ mg) was also orally administered in doses of 50 mg/kg and 10 mg/kg. After definite inter vals, the rats were anesthetized with urethane (2 g/kg i.p.) and then sacrificed. The intesti nal preparation was washed in water and a part of ileum was excised and used for the ex periment. As the muscle specimen the quadriceps was used. Samples from various tis sues weighing from 15 to 25 mg wet weight were transferred to counting vials. One ml of NCS solubilizer was added to the samples and left standing at room temperature for about 24 hr. After solubilization, 10 ml of toluene scintillator solution was added to the samples and left standing in a dark place for l hr and thereafter was counted. Five ml of methanol were added to 0.5 ml of plasma and centrifuged (500 x g, 10 min) after being left standing for 30 min at room temperature. One ml of supernatant was transferred to a counting vial containing 10 ml of dioxane scintillator solution and counted. The urine sample was diluted twenty times and I ml aliquot was transferred to a count ing vial containing 10 ml of dioxane scintillator solution and counted. Control urine sam ples were used as blanks. All samples were counted for three, one-minute periods. Chromatography: Paper chromatography was carried out by using Whatman No. 1 paper and n-butanol-acetic acid-water (4: 1: 5) as the solvent. Urine specimens were collected for 24 hr after intraperitoneal administration of metaproterenol at a dose of 500 mg/kg in rats. Q-glucuronidase was added to urine samples at a concentration of 30 mg/ml. The mixture was incubated for 1 hr at 40°C. Aliquots of urine hydrolysed with ~-glucuronidase and aliquots of original urine were evaportated and column chro matography was carried out using Dowex 50W x 4[W]. The column was first washed with 5 ml of water and then with 5 ml of 2N-NH4OH. The fraction eluated with 2N NH4OH was evaporated and chromatographied on paper as described above. Radioana1vsis: The radioactivity assays were performed in an Aloka LSC 501 liquid scintillation spectrometer. The toluene scintillator solution contained 4 gr of DPO [2, 5-diphenyloxazole], 0.1 gr of POPOP [p-bis 2-(5-phenyloxazolyl)-benzene] in I liter of toluene. The dioxane scintillator solution contained 6 gr of DPO, 0.275 gr of POPOP and 112 gr of naphthalene in I liter of dioxane. Samples were counted in glass vials with a diameter of 25 mm and a capacity of 20 ml. RESULTS 1. Plasma level: The 3H-radioactivity level in rat plasma showed its maximal value in 1 hr after 3H-metaproterenol in a dose of 50 mg/kg i.p. The plasma level was linearly lowered down to 40% of the maximal value 5 hr after injection. After 24 hr, 10;•() of the maximal plasma level was still detected. The biological half life determined from the plasma radio activity disappearance curve, was 4 hr and 30 min. Fic. 1. Plasma radioactivity decay after admi nistration of 3H-metaproterenol to rats. After oral administration of 3H-meta proterenol in a dose of 50 mg/kg, the plasma level of radioactivity reached maxi mum in I hr, and was slowly reduced by 60% within 3 hr and then slightly re-elevated after 5 hr. A relatively high level of radioactivity still remained after 24 hr following oral administration. The maximal plasma level of radioactivity in oral administration, was approximately one third in height as compared with that of intraperitoneal injection. However, the rate of decrease of the plasma level was much slower in the case of oral administration. 2. Tissue distribution: The 3H-radioactivity distribution in various tissues was investi gated 1 hr after i.p. injection of 3H-metaproterenol in a dose of 50 mg/kg. A higher level of radioactivity was observed in the kidneys and liver than any other tissues and was main tained at a relatively high level 5 hr after administration. Radioactivities observed in the intestines, lung and heart were considerable but such was very low in muscles and almost negligible in the brain. Radioactivity was still observed in various tissues at 24 hr after 3H-metaproterenol administration (Table 1). As shown in Table 2, radioactivity distributed in various tissues was not markedly decreased at 24 hr after oral administration of 3H-metaproterenol. Thus, it was noted that the 3H-radioactivity in various tissues after p.o. administration decreases more slowly than that in the case of i.p. administration. TABLE 1. Tissue distribution of 3H-radioactivity in rats following of 3H-metaproterenol (50 mg/kg i.p.) (3H-metaproterenol : sp. act. 14 tec/mg) TABLE. 2. Tissue distribution of 3H-radioactivity in rats following of 3H-metaproterenol (10 mg;~kg p.o.) (3H-metaproterenol : sp. act. 16 pcjmg) TABLE. 3. "Twenty-four hr excretion of tritium in rat urine following 3H metaproterenol (50 mg/kg p.o.) Fic. 2. Paperchromatogram of rat urine collected fractionally following metaproterenol admini stration, FIG. 3. Paperchromatogram of synthetic refe rence compounds. VMA : vanillyl mandelic acid 3,4 DMA : 3,4-dihydroxy mandelic acid MN : metanephrine NMN : normetanephrine 3. Urinary excretion: After 3H-metaproterenol was orally administered in a dose of 50 mg/kg to rats, 24 hr urine was collected to measure total elimination. As shown in Table 3, 40„% of the administered 3H-radioactivity was eliminated in the urine specimens collected during 24 hr following 3H-metaproterenol administration. 4. Metabolites excreted in the rat rtrine: Rat urine was collected fractionally every 8 hr for 24 hr after the administration of non-labeled metaproterenol given orally in a dose of 500 mg/kg. Each urine specimen was used for identifying amine components excreted in the urine by paper chromatography. The qualitative identification was carried out by using a Folin-Chiocalteu regent. The first and second urine specimens showed the same pattern in chromatograms, in which unchanged metaproterenol was distinctly identifi able. In the third urine specimen, however, unchanged metaproterenol was not identified in 9 out of the I I cases. These urine specimens were chromatographied again after being treated with (l-glucuronidase. As illustrated in Fig. 2, treatment with Q-glucuronidase involved the appearance of a distinct metaproterenol spot on paper chromatograms in 3 out of 9 cases in which, otherwise, no metaproterenol spot was identified. It was also confirmed that metanephrine, normetanephrine, 3, 4-dihydroxy mandelic acid vanillyl mandelic acid and dopamine were negative in all urine specimens used in the present ex