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Pharmacokinetic and Metabolic Studies of Higenamine in Rabbits
((+/-)-Higenamine, (+/-)- HG), a potent cardiotonic principle,
is isolated as a racemate from Aconitum japonicium, and other
natural resource such as Annona squamosa, Asiasarum
heterotropodes, and Gnetum parvifolium. It acts directly on
the adrenergic beta-1 and beta-2 receptors. (+)-HG is isolated
from Nelumbo nucifera as smooth muscle relaxant. A method for
the pharmacokinetic studies of HG in plasma and urine based on
high - performance liquid chromatography (HPLC) with
electrochemical detection was developed. The plasma and urine
was treated with acidic alumina and then HG was released by acid
treatment. HPLC was performed on an ODS column with a mobile
phase of acetonitrile-0.1 % phosphoric acid (9:91) and
electrochemical detector at an oxidation potential of0.75 V.
The lower limits of quantitation for HG in plasma and urine was
2.645 and 10.58 ng/mL, respectively. The recoveries of HG after
alumina treatment in plasma and urine were ca. 77.5 and 84.4 %,
respectively. Intra- and Inter-day precision and accuracy
reported as coefficients of variation in plasma and urine
were less than 7 %.
The pharmacokinetics of HG were investigated in rabbits by iv
bolus ( 10, 20,30 mg/kg), peroral route (50 mg/kg) and iv
infusion (107 ug/min/kg). Plasma HG concentration declined
rapidly in a biexponential pattern, with a terminal half -life
of 22 min.The AUC increased proportionally with increasing
doses, whereas the percentage of unchanged HG excreted from
urine remained constant although dose was increased. The mean
of total plasma clearance, renal clearance, mean residence time,
volume of distribution at steady-state, and fraction of urinary
excretion were 127.7 mL/min/kg, 6.9 mL/min/kg, 9.28 min, 1.44
L/kg and 5.48 %, respectively. The mean percentage of protein
binding of HG in plasma was 54.8 % at steady-state after iv
infusion. The results from post-infusion were also confirmed
that HG displayed a two-compartment open model in animals.
After oral administration,HG was rapidly absorbed to reach peak
concentration within 10 min. Interestingly, the plasma
concentration-time profiles revealed two distinguishable groups
with different Cmax,extent of absorption and urinary excretion.
The average absolute bioavailabilities of HG calculated by AUCs
and corrected with renal clearance were about 20.5 % and 5.5 %
for the two groups, respectively.
Upon hydrolysis of urine samples with beta -
glucuronidase, urinary concentrations of HG were greatly
enhanced in both groups in spite of the administration
routes. Parent drug and its conjugated excreted from urine was
about 20 - 40 % of the doses. After iv bolus of HG.HCl at 20
mg/kg to a rabbit , HG was mainly excreted as conjugated
metabolites from bile about 6 %. The acute toxicity test was
intravenously performed on mice, the LD50 was about 50 mg/kg
with no sex difference. Using the column chromatography (MCI
gel) and preparatived HPLC (RP-C18) with photodiode array
detector, at least 8 distinct urinary metabolites from the 24 h
pooled urine sample after oral administration were isolated as
crystalline solids. Metabolites and its derivatizations were
characterized using a combination of negative-ion LC/MS, and
2DNMR spectroscopy to determine the conjugation position.
After acid hydrolysis of metabolites, the configuration of each
optical aglycone of HG metabolites was determined by using a
beta-cyclodextran chiral column and compared with the optically
active HG enantiomers. One metabolites was identified as
S-(-)-HG-6,7-O-beta-D- diglucuronide (M1). Six metabolites were
HG mono glucuronides, S-(-)-HG-13-O- beta-D-glucuronide (M2),
R-(+)-HG-7-O-beta-D-glucuronide (M3), R-(+)-HG-13-beta -D-
glucuronide (M4), S-(-)-HG-6-O-beta-D-glucuronide (M5), R-(+)-
HG-6-O-beta-D- glucuronide (M6) and S-(-)-HG-7-O-beta-D-
glucuronide (M7). Metabolite 8 was determined as S-(-)-HG-7-
O-sulfate (M8). Based on the HPLC profiles, the major formation
of conjugation were glucuronidation occurred at the C-6 and
C-7 -OH about 40 % and 45 %, respectively, but minor
glucuronidation at the C-13-OH (ca 3 %), sulfation and
methylation via COMT. Interestingly, there are marked
differences in stereoselective glucuronidation between the
enantiomers of HG at the catechol moiety. The conjugated
ratios of R-(+)/S-(-) isomer of HG at the C-6 and C-7-OH were
about 3 and 0.1, respectively. The peak area ratios of HG
metabolites indicated that (+/-)-HG was present of regio- and
enantioselective metabolism in rabbits.
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