Pharmacokinetics on plasma and blood cells concentration, tissue distribution and excretion of amrubicin hydrochloride (SM-5887), a novel antitumor agent, were investigated after a single intravenous administration of ¹⁴C-labeled SM-5887 at the doses of 0.1, 1 and 10 mg/kg to rats.
1. The radioactivity levels in plasma and blood cells at these doses decreased multi-exponentially, and the levels of radioactivity in blood cells were 1/2 ?? 1/3 of those present in plasma. The time course profiles were almost parallel among these doses (0.1 ?? 10 mg/kg), and the linear relationship in the area under the concentration time-curve and administered doses was observed.
2. Plasma and blood cell concentrations of the unchanged SM-5887 at these doses decreased more rapidly than those of total radioactivity, and the levels in blood cells were about a half of those in plasma. The α, β and γ-phase half-lives of SM-5887 concentration in plasma were 1.8 min, 29 min and 1.9 hr, respectively.
3. The concentrations of amrubicinol (SM-5887-13-OH), the major bioactive metabolite, in plasma and blood cells increased rapidly, and then decreased slowly. In blood cells, the level of SM-5887-13-OH was three times as high as that in plasma. Other aglycone metabolites without the sugar moiety were also detected in plasma and blood cells.
4. The radioactivity level was relatively high in such tissues as bone marrow, intestinal wall, skin, adrenal, spleen, lung, Harderian gland, submaxillary gland, kidney and liver at 1 ?? 4 hr after administration. Then the radioactivity disappeared gradually from most tissues in a similar profile as that in plasma, except a few tissues such as testis, submaxillary gland, thymus and hair.
5. Within 168 hr after administration, 13% and 76% of the dosed radioactivity was excreted into urine and feces, respectively. Within 72 hr after administration, 58% of the dosed radioactivity was excreted into bile. After intraduodenal injection of the bile to the bile-duct cannulated rats, 31% of the injected radioactivity was reabsorbed.
6. Little difference in pharmacokinetic profile was observed between male and female rats.
7. In vitro protein binding of SM-5887 and SM-5887-13-OH in mammalian plasma and HSA were 92 ?? 97% and 78 ?? 93%, respectively. In vivo plasma protein binding of SM-5887 and SM-5887-13-OH after intravenous administration of SM-5887 to rats were 93 ?? 96% and 82 ?? 89%, respectively.

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Pharmacokinetics in plasma and blood cells concentrations, tissue distribution and excretion of amrubicin hydrochloride (SM-5887), a novel antitumor agent, were investigated during and after repeated intravenous administration of ¹⁴C-labeled SM-5887 at a dose of 1 mg/kg/day to rats for 14 days.
1. The radioactivity levels in plasma and blood cells at 24 hr after each dosing during repea ted administration increased with the number of dosing, and the concentrations after the 14th dosing were 6 ?? 8 times higher than those after the first dosing. The AUC_0-168h after the 14th dosing was almost 5 times as large as that after the first dosing. These concentrations during repeated administration can be simulated closely from the time-course profile after the first dosing.
2. The concentrations of SM-5887 and its metabolites in plasma and blood cells after the 14th dosing were similar to those after the first dosing.
3. The radioactivity levels in the tissues after the 14th dosing were relatively high in the submaxillary gland, Harderian gland, liver, kidney, adrenal and pituitary. The radioactivity disappeared gradually from most tissues, especially from submaxillary gland, testis and hair.
4. In the tissues, the concentrations of SM-5887 after the 14th dosing were high in submaxillary gland, spleen, lung, thymus and bone marrow. Amrubicinol (SM-5887-13-OH) concentrations were high in sub maxillary gland, thymus and adrenal. Met B concentrations were high in Harderian gland, submaxillary gland, adrenal and kidney. The ratios of the unextracted radioactivity, that was probably attributed mainly to polar metabolites, in plasma, liver, and bladder, were relatively high.
5. The excretion ratios of radioactivity into urine and feces with in 168 hr after 14th dosing, were 14% and 80% of the total dosed radioactivity, respectively. This result was similar to that after a single dosing.

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Pharmacokinetics in plasma and blood cells concentrations, tissue distribution and excretion of amrubicin hydrochloride (SM-5887), a novel antitumor agent, were investigated after a single intravenous administration of ¹⁴C-labeled SM-5887 at a dose of 1.5 mg/kg to dogs.
1. The radioactivity levels in plasma and blood cells decreased multi-exponentially, and the levels of radioactivity in blood cells were 1/2-1/3 of those in plasma.
2. The concentrations of the unchanged SM-5887 in plasma and blood cells decreased more rapidly than total radioactivity. The α- and β-phase half lives of SM-5887 concentration in plasma were 20 min and 3.8 hr, respectively. Plasma and blood cell concentrations of amrubicinol (SM-5887-13-OH), the major bioactive metabolite, increased rapidly, and then decreased slowly. Other aglycone metabolites without the sugar moiety were also detected in plasma and blood cells.
3. The radioactivity in each tissue reached a maximum concentration at 1 or 4 hr after administration. The radioactivity levels were relatively high in most tissues including liver, gallbladder, kidney, lung, spleen, pancreas and prostate gland. The radioactivity in most tissues disappeared gradually with a profile similar to that in plasma, except few tissues such as liver, eye, pigmented skin and testis. In liver, 9 % of the dosed radioactivity was observed at 168 hr after administration.
4. The major metabolites in tissues were Met B and SM-5887-13-OH. The concentration of SM-5887 in tissues were low, and decreased rapidly.
5. Within 168 hr after administration, 8 % and 74% of the dosed radioactivity were excreted into urine and feces, respectively.

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The metabolism of amrubicin hydrochloride (SM-5887), a novel antitumor agent, was investigated in rats and dogs after intravenous administration of ¹⁴C-labeled SM-5887.
1. Nine metabolites in rat urine, ten metabolites in rat bile, six metabolites in dog urine and eleven metabolites in dog bile were observed after intravenous administration of ¹⁴C-SM-5887. Major radioactive components in rat urine were amrubicinol (SM-5887-13-OH) and the unchanged SM-5887, and those in dog urine were M-2 and Met B. Major radioactive components in bile of rats and dogs were polar metabolites, such as M-1 and M-2.
2. In rat tissues, ma jor radioactive components were nonpolar or less polar metabolites, such as Met B and SM-5887-13-OH, and the unchanged SM-5887.
3. The structures of the nonpolar or less polar metabolites (SM-5887-13-OH and Met A ?? Met D) were determined by comparing them to the authentic samples using HPLC and mass spectrometry. The identified metabolites were an alcoholic derivative (SM-5887-13-OH), deoxyaglycones (Met A and Met B) formed by reductive glycosidic cleavage, an aglycone (Met B) formed by glycosidic cleavage and a deaminated derivative (Met D). Two polar metabolites (M-4 and M-6) were also identified as the conjugates of SM-5887-13-OH with glucuronic acid. The other polar metabolites (M-1 ?? M-3, M-5 and M-7), which were assumed to be conjugates based on their high polarities, could not be identified due to their low stabilities.

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To determine human cytochrome P450 isoform(s) (CYPs) involved in the metabolism of pranidipine, a new potent and long-acting 1, 4-dihydropyridine calcium antagonist, the biotransformation of the compound was investigated in vitro using ten isoforms of human cytochrome P450 expressed in human AHH-1 TK +/- cell lines. Modulation of an ethyl acetate-extract of grapefruit juice on the activity of CYP3A4-mediated pranidipine metabolism was also investigated.
1. Pranidipine was dehydrogenated into pyridine metabolite by CYP3A4 with Km and V_max values of 21.4 μM and 0.110 nmol/min/mg protein, respectively. CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 2E1 were not involved in the pranidipine metabolism.
2. MOP-13031 constructed with dihydropyridine ring, a metabolite of pranidipine, was also oxidized into pyridine ring by the multi-isoforms of cytochrome P450 including CYP3A4, 2C19, 2D6 and 2E1. The Km and V_max values on the oxidation of MOP-13031 to pyridine metabolite by CYP3A4 were 796 μM and 0.124 nmol/min/mg protein, respectively.
3. CYP3A4-mediated testosterone 6β-hydroxylase activity was competitively inhibited by pranidipine with a Ki value of 5.44 μM but was not affected by MOP-13031 up to a concentration of 100 μM. (±) Miconazole inhibited the hydroxylation up to 94.7% at 10 μM.
4. CYP3A4-mediated pranidipine dehydrogenase activity was dose-dependently decreased by an addition of the ethyl acetate-extract of grapefruit juice.

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Plasma levels of radioactivity, the unchanged drug and major metabolites were investigated in rats after a single oral or intravenous administration of ¹⁴C-BX661A (labeled at 5ASA moiety: ¹⁴C-BX661A (5ASA), labeled at 4ABA moiety: ¹⁴C-BX661A (4ABA)) and non-labeled BX661A.
1. After oral administration of ¹⁴C-BX661A to fasted rats, plasma levels of radioactivity showed 2 peaks with maximum time (T_max) at 30 min and 8-10 hr. Concentrations of the 1st peaks were same in both labeled compounds, however those of 2nd peaks were different from each other, plasma concentrations at 8-10 hr were higher in ¹⁴C-BX661A(5ASA) and those at 24 hr were higher in ¹⁴C-BX661A(4ABA).
2. Acetylation of 5ASA was saturated at the high dose, and that of 4ABA was saturated at the low dose.
3. After oral administration of BX661A to fasted female rats, plasma concentrations of the unchanged drug were lower than those of male rats.
4. After oral administration of BX661A to non-fasted male rats, concentrations of the unchanged drug, 5ASA and Ac 5ASA were lower than those of fasted rats.On the other hand, plasma concentration of 4ABA and Ac-4ABA were not influenced by diet.
5. After intravenous administration of BX661A to male rats, the unchanged drug was declined with T_1/2(α) of 6.0-6.2 min and T_1/2(β) of 21.8-28.6 min. On the other hand, metabolites were not detected during the first 2 hr.
6. After intravenous administration of BX661A to female rats, significant differences were observed in T_1/2(α) (male: 6.2 min, female: 3.4 min), K_el (male: 0.068/min, female: 0.143/min), CL (male: 0.015 l/min/kg, female: 0.025 l/min/kg) and AUC_{(0-2 hr)} (male: 179744 ng·/ml, female: 111370 ng·h/ml).

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The influence of diarrhea, which was the prime symptom of the ulcerative colitis, on pharmacokinetics of BX661A was investigated using DSS-induced ulcerative colitis model in rats.
1. After oral administration of ¹⁴C-BX661A or BX661A to model rats, plasma transitions of radioactivity and each compounds were similar to those of normal rats. Pharmacokinetics parameters also showed similar values.
2. After oral administration of BX661A to normal and model rats, the behavior of BX661A in gastrointestinal tract was similar in both rats. After administration of BX661A, the metabolites were hardly detected in the stomach and small intestine and more than 80% of the dose existed as unchanged drug at 2 hr after dosing. Whereas in caecum, the unchanged drug was recognized slightly, and 5-ASA, Ac-5-ASA, 4-ABA and Ac-4-ABA were abundant for the first 6-14 hr. In the large intestine, the unchanged drug was not detected over the observation time, and the contents of metabolites were maximal for the first 10 ?? 14 hr.

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The distribution and excretion of radioactivity, and of the unchanged drug and major metabolites were investigated in rats after a single oral or intravenous administration of ¹⁴C-BX661A (labeled at 5ASA moiety: ¹⁴C-BX661A (5ASA), labeled at 4ABA moiety: ¹⁴C-BX661A (4ABA)) and non-labeled BX661A.
1. After oral administration of ¹⁴C-BX661A(5ASA) to fasted male rats, the radioactivity reached the highest level at 10 hr after administration in most tissues, except gastrointestinal tract. At this time point, the tissues showing higher level of radioactivity than that in plasma, were liver and kidney, except gastrointestinal tract. After administration of ¹⁴C-BX661A (4ABA), similar results were obtained. However, the levels of radioactivity were lower than those of ¹⁴C-BX661A (5ASA).
2. During 72 hr after oral administration of ¹⁴C-BX661A (5ASA) to fasted male rats, about 32 and 66% of the dose were excreted into the urine and feces, respectively. In female rats, about 45 and 55% of the dose were excreted into the urine and feces, respectively. While in ¹⁴C-BX661A (4ABA), the excretion of radioactivity into the urine and feces were about 7 and 88% of the dose in male rats, and the similar results were obtained in female rats.
3. During 72 hr after oral administration of BX661A to fasted male rats at a dose of 50, 100 and 200 mg/ kg, the excretion of the unchanged drug into the urine and feces were less than 0.6% of the dose. It was suggested that azo linkage of BX661A was almost completely cleavaged. Urinary excretion of 5ASA and Ac-5ASA accounted for 0.0-2.2% and 21.1-24.5%, and fecal excretion were 22.7-25.3% and 20.0-32.1% of the dose, respectively. On the other hand, urinary excretion of 4ABA and Ac-4ABA were less than 0.8 and 3.4-4.3% of the dose, respectively. Fecal excretion of 4ABA and Ac-4ABA were 67.3-78.5% and 2.7-5.1% of the dose, respectively.
4. During 72 hr after oral administration of BX661A to fasted female rats at a dose of 100 mg/kg, the fecal excretion of Ac-5ASA was significantly higher than those of male. Excretion pattern of another compounds were similar to those of male rats.
5. During 72 hr after intravenous administration of ¹⁴C-BX661A(5ASA) to fasted male rats, about 43 and 55% of the dose were excreted into the urine and feces, respectively. After administration of ¹⁴C-BX661A (4ABA), about 29 and 66% of the dose were excreted into the urine and feces, respectively.
After intravenous administration of BX661A to fasted male rats, 29.5% of the dose was excreted into the urine as the unchanged drug within 8 hr. On the other hand, urinary excretion of Ac-5ASA was 11.5%, and fecal excretions of 5ASA, Ac-5ASA and 4ABA were 4.9, 19.3 and 29.0% of the dose during 72 hr, respectively.
6. After oral administration of ¹⁴C-BX661A to fasted male rats, the biliary excretions within 24 hr were 3-4% of the dose in both labeled compounds.
After intravenous administration of ¹⁴C-BX661A (5ASA) to fasted male and female rats, the biliary excretion within 120 min was low in male rats compared with female (male: 67%, female: 80%).
7. Azo-bond of BX661A was easily reduced by reacting with human feces. After 90 min of reaction, the unchanged drug was not detected and the formation of 5ASA was observed. However, Ac-5ASA was not detected in the reaction mixture.

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Pharmacokinetics of BX661A was investigated following 7-day or 14-day period of daily oral administration of ¹⁴C-BX661A(¹⁴C-BX661A(5ASA): labeled at 5ASA moiety, ¹⁴C-BX661A(4ABA): labeled at 4ABA moiety) and non-labeled BX661A to male rats.
1. Two metabolites, acetyl-p-aminobenzoic acid and acetyl-p-amino-hippuric acid were found in the urine.
2. After repeated oral administration of ¹⁴C-BX661A(5ASA) to rats, no differences were observed in C_max, AUC and T_max between the 1st, 7th and 14th dose. While after repeated oral administration of ¹⁴C-BX661A(4ABA) to rats, plasma level of radioactivity at 24 hr after the 7th dose was increased to more than 15 times of the 1st dose. But C_max, AUC and T_max of 14th dose were almost equal to those of 7th dose.
3. The plasma transition of BX661A and its metabolites did not change during 14-day period of daily oral administration of BX661A. At 24 hr after every dosing, plasma concentrations of BX661A and its metabolites were not detectable or were close to the limit of detection.
4. After repeated oral administration of ¹⁴C-BX661A(5ASA) to rats, tissue radioactivity levels at 24 hr after the 7th dose increased to about 2 times of the 1st dose, but the levels after the 14th dose were almost equal to those of 7th dose. After the final dose, the radioactivity in all tissues was decreased slowly and the accumulation was not observed.
5. After repeated oral administration of ¹⁴C-BX661A(4ABA) to rats, tissue radioactivity levels at 24 hr were increased with dosing. However, the ratios of tissue/plasma concentration were unchanged in all tissues tested. After final dose, the elimination of radioactivity from all tissues were slower than that of plasma.
6. The excretion pattern of BX661A and its metabolites into urine and feces were almost constant during the repeated oral administration.

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For the analysis of antipyretics, it was assumed that: (1) The rat body is divided into two compartments, core and skin. (2) Metabolic heat (M) is generated in the core compartment. (3) Heat loss by vaporization (V) is mainly respiratory effect and occurs in the core compartment. (4) At the skin compartment, heat is gained from the core compartment by conduction (K) and is transferred to the ambient air by radiation and convection. (5) Central nervous system commands the efferent signals for M, K and V to change their values according to the changes in afferent signals from core and skin temperature. (6) The effect of antipyretics acts as afferent signals to the controller. For loop diuretics, it was assumed that: (1) The diuretic rate can be correlated with the urinary excretion rate of the drug. (2) If there is no intervention of the body fluid regulation system, the relationship of the diuretic rate and the corresponding urinary excretion rate can be described by Hill equation. (3) Intensity of the body fluid regulation is also described by Hill equation, in which, the intensity is correlated with the cumulative amount of the drug excreted in urine. For neuromuscular blockade, assumptions were: (1) There exists an acetylcholine (ACh) compartment at a motor nerve terminal. (2) ACh in the compartment is eliminated by a first-order rate process. (3) All of the ACh in the compartment is released by one electrical stimulus. (4) The compartment is replenished by two kinds of ACh mobilization. One is a slow mobilization with a constant rate and the other is a momentary mobilization which takes place just after the release of ACh. (5) The released ACh is metabolized immediately after binding to receptors and causing twitch response.

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The properties of cytochrome P450 varies among molecular forms over animal species. Thus, such a variation is believed to result in the species differences in drug metabolism. We have performed research to predict drug metabolism in humans. We purified and cloned forms of cytochrome P450 from animals and humans, and clarified the properties by expression in hetelrologous system. Among them, CYP3A7 is a form of cytochrome P450 present specifically in human fetal livers. First, we analyzed 5'-upstream region to know elements involved in the respective fetal and adult specific expression of CYP3A7 and CYP3A4. We found that there were two elements which might cause the fetal and adult specific expression of these cytochromes. To answer a question if cytochrome P450 acts in vivo as estimated by in vitro studies, we established transgenic mice carrying CYP3A7 gene. When the transgenic mice were treated with aflatoxin B₁, DNA damage as well as aflatoxin B₁ DNA adduct occurred only in organs in which CYP3A7 was expressed.

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The objectives of pharmacokinetics and metabolic studies in drug safety evaluation is the drug absorption, the duration of drug exposure and the effects of multiple dose regimen on the pharmacokinetics in test animal species. There are many examples of drug to show little or no toxic event at extremely high dose level, which originate in low absorption or low bioavailability of the drug. A complicating situation can arise when the pharmacokinetic profile of the drug changes during repeated dosing due to such factors as enzyme induction, saturation of metabolism, accumulation of drug in the tissue, and renal excretion process. The toxic effect could also changed their pharmacokinetics during a toxicity testing. It is important to monitor a drug level during the toxicity testing. When toxicity cause depends on toxic metabolites or reactive intermediates, the plasma concentration of unchanged drug are difficult to correlate to the toxic efect. It is indispensable to evaluate the metabolic pathways and metabolic clearance of a durg under similar conditions of toxicity testing. These data may be useful for the interpretation of toxicological finding and their relevance to clinical safety evaluation.
Stereoselective pharmacokinetics and metabolic studies should be evaluated because of differences on the pharmacokinetics and metabolism of each enantiomer. Most of dihydropyridine calcium antagonists have one or more chiral centre, and the pharmacological activity between the enantiomers for these durg is known to be markedly different. The stereoselective pharmacokinetics of these drugs in animals, healthy subjects and patients should evaluated during the development process. Enantiomer-enantiomer interaction, enantiomeric inversion and the stereochemical aspects of pharmacokinetic drug interactions in these drugs should also be studied.

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Bile acids are synthesized from cholesterol in the liver excreted into duodenum via the bile duct as their glycine and taurine conjugates, and converted in part into secondary bile acids by intestinal bacteria. It is well-known that further hydroxylation at C-1, -2, -4, -6 and -19, and conjugation at C-3 with glucuronic acid or sulfuric acid take place in the liver. In this paper bio-analytical studies on bile acids by hyphenated mass spectrometry related to drug metabolism will be discussed.
In the first, to clarify an extrahepatic reduction system, ¹⁸O labeled 3-oxo bile acids were synthesized and after incubation with human blood, biotransformed products were separated and characterized by GC-MS. The 3-oxo group was reduced into the 3α- and 3β-hydroxyl function catalyzed by the enzyme (s) in human red blood cells. This result strongly implies that biologically active compounds such as drugs with the oxo group are also transformed into hydroxylated metabolites in human blood.
On the biosynthesis of bile acids, the final step involving the oxidative cleavage of a side chain of 5β-cholestanoic acid, which has a chiral center at C-25, to form primary bile acids takes place in the peroxisomes. From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 5β-cholestanoic acid into (24E)-5β-cholest-24-enoic acid was then studies with GC-MS. Substrates labeled with ²H at C 24 and C-25 stereoselectively were incubated with a rat liver light mitochondrial fraction and the stereospecific elimination of a pro-R hydrogen at C-24 in both (25R) and (25S)-cholestanoic acids indicating syn-elimination for the former, whereas anti-elimination for the letter was observed. When CoA thioesters of (25R)-and (25S)-5β-cholanoic acids were incubated individually with a rat liver peroxisomal fraction, rapid epimerization to form an equal mixture of stereoisomers from either direction was occurred. Moreover only the 25S antipode was easily transformed into dehydrogenated 5β-cholestanoic acid by peroxisomal acyl-CoA oxidase.
The conjugation of carboxylic acids with n-glucuronic acid to form acyl glucuronides plays a significant role in the metabolism and disposition of drugs. The formation of 24-acyl glucuronides of bile acids in the rat liver are also discussed.

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