Effects of 4-hydroxyantipyrine and its 4-O-sulfate on antipyrine as biodistribution promoter

Article abstract of DOI:10.1248/bpb.24.529

The effects of 4-hydroxyantipyrine (4-OH), a major metabolite of antipyrine, and its 4-O-sulfate (4-S) on the pharmacokinetics of antipyrine were investigated in rats. Plasma elimination of intravenously administered antipyrine was significantly decelerated under a steady-state concentration of 4-OH but not under that of 4-S. Tissue-to-plasma concentration ratio (K_p) of antipyrine under its steady-state concentration was significantly increased in the brain and heart by the concomitant use of 4-OH, while similar use of 4-S had no effect. The enhancement of the blood-brain barrier (BBB) permeability of antipyrine caused by the concomitant use of 4-OH was believed to be concerned with the increase of the K_p value of antipyrine in the brain. These results suggested that 4-OH could be used as a biodistribution promoter.

Full text of DOI:10.1248/bpb.24.529

May 2001
Biol. Pharm. Bull. 24(5) 529—534 (2001)
529
Effects of 4-Hydroxyantipyrine and Its 4-O-Sulfate on Antipyrine as
Biodistribution Promoter
Yuhsuke OHKAWA, Miki MATSUMURA, Yuhji KUROSAKI, Masateru KURUMI, Kenji SASAKI, and
*
Taiji NAKAYAMA
Faculty of Pharmaceutical Sciences, Okayama University, 1–1–1 Tsushima-naka, Okayama 700–8530, Japan.
Received March 1, 2000; accepted January 13, 2001
The effects of 4-hydroxyantipyrine (4-OH), a major metabolite of antipyrine, and its 4-O-sulfate (4-S) on the
pharmacokinetics of antipyrine were investigated in rats. Plasma elimination of intravenously administered an-
tipyrine was significantly decelerated under a steady-state concentration of 4-OH but not under that of 4-S. Tis-
sue-to-plasma concentration ratio (Kp) of antipyrine under its steady-state concentration was significantly in-
creased in the brain and heart by the concomitant use of 4-OH, while similar use of 4-S had no effect. The en-
hancement of the blood-brain barrier (BBB) permeability of antipyrine caused by the concomitant use of 4-OH
was believed to be concerned with the increase of the Kp value of antipyrine in the brain. These results suggested
that 4-OH could be used as a biodistribution promoter.
Key words biodistribution promoter; antipyrine; 4-hydroxyantipyrine; 4-hydroxyantipyrine O-sulfate; concomitant use;
drug/metabolite interaction
Considerable attention1—4) has been focused on drug/drug
(150 ml). The ammonia gas was bubbled into the solution for
1.5 h and precipitated solid was filtered off. The filtrate was
concentrated to ca. one-third under reduced pressure at
50 °C. Diethyl ether was then added to make the solution a
milky suspension and allowed to stand in a refrigerator. The
precipitated crystalline solid was collected by filtration to
give 20 g (65%) of the titled compound as an ammonium salt
which gave a single spot on tlc in several solvent systems.
The structure of this compound was consistent with instru-
mental data and analysis data, mp 210—214 °C (decom.).
Anal. Calcd for C11H15N3O5S: C, 43.85; H, 5.02; N, 13.95.
Found: C, 43.61; H, 5.27; N, 13.79.
interactions such as those in drug distribution, drug metabo-
lism, drug absorption and receptor binding in multiple-drug
therapy. In addition to drug/drug interactions, in the 1960s
we reported the significance of drug/metabolite interaction(s)
in the biopharmaceutical fate of a drug.5—10) Studenberg and
Brouwer also reported that the metabolites of phenobarbital
inhibited the biotransformation of acetaminophen.11) More
recently we reported the effects of the concomitant use of N-
acetyl-p-aminophenyl O-sulfate (APAPS)12—14) and its geo-
metric isomers.15) In those reports, it was found that the tis-
sue-to-plasma concentration ratio (Kp) of acetaminophen was
significantly increased in the liver, heart, kidney and brain by
the presence of APAPS but not by its geometric isomers.
That is, APAPS seemed to play a role as a biodistribution
promoter. This result prompted us to examine whether a sim-
ilar drug/metabolite interaction could be observed when
other drugs and metabolites were employed.
Animals Male Wistar rats (220—320 g) (Japan SLC,
Inc., Hamamatsu, Japan) were used.
In Vivo Experiments The rats were fasted overnight be-
fore use, but allowed free access to water, and were fixed in a
dorsal position under urethane anesthesia (900 mg/kg, i.p.).
Polyethylene tubing was cannulated into both the left femoral
vein and the right femoral artery.
Pharmacokinetics of Antipyrine, 4-OH and 4-S: An-
tipyrine in saline (50 mg/kg), 4-OH in 10 mM NaOH–saline
(50 mg/kg) and 4-S in saline (50 mg/kg) were respectively in-
travenously administered through the right femoral vein in a
volume of 1 mg/kg, respectively. A blood sample was col-
lected through the cannula fastened to the femoral vein at ap-
propriate time intervals. Plasma samples were obtained by
centrifugation at 10000ϫg for 2 min, and all plasma samples
were stored at Ϫ20 °C until analyzed.
Effect of 4-OH and 4-S on Plasma Concentration–Time
Curves of Antipyrine: 4-OH and 4-S were intravenously ad-
ministered at the initial dose (4.40 mg/kg and 6.13 mg/kg, re-
spectively), followed by constant infusion (52.9 mg/kg/h and
44.2 mg/kg/h, respectively) to maintain the target plasma
concentration (10 mg/ml and 50 mg/ml, respectively) through
the cannula fastened to the left femoral vein throughout the
experiment. Antipyrine (50 mg/kg) was intravenously admin-
istered under a steady-state concentration of 4-OH (or 4-S)
120 min after the start of infusion, and 10 min later, blood
was continually collected from the right femoral artery.
Saline was used as a control.
In this report, the possibility of 4-hydroxyantipyrine (4-
OH),16) which is one of the major metabolites of antipyrine,
and its 4-O-sulfate (4-S) as a biodistribution promoter was
examined by their concomitant use with antipyrine which is
used as a bioactive drug.
MATERIALS AND METHODS
Materials 4-OH and 4-S (as ammonium salt) were pre-
pared in our laboratory as below. Antipyrine and 4-S were
dissolved in saline. 4-OH was dissolved in 10 mM NaOH
containing saline before use. All chemicals and reagents
were commercially available and of analytical grade or better.
Preparation of 4-OH This compound was prepared by
the method of Hukki and Myry16) to afford colorless needles
(74%), mp 193—195 °C (lit.16) 182 °C).
Preparation of 4-S To the solution of 4-OH (20 g, 0.10
mmol) in pyridine (300 ml) was added pyridine-sulfur triox-
ide complex (30 g, 0.19 mol) and the mixture was stirred at
0—10 °C for 48 h. After evaporation of the solvent of the re-
action mixture in reduced pressure at 50 °C for 50 min. The
resulting pale yellow viscous oil was dissolved in MeOH
To whom correspondence should be addressed. e-mail: nakayama@pheasant.pharm.okayama-u.ac.jp
© 2001 Pharmaceutical Society of Japan

530
Vol. 24, No. 5
Effect of Bolus Challenge with 4-OH and 4-S on Plasma and the aqueous layer was diluted with purified water. The
Concentration Profile of Antipyrine: Antipyrine was intra- concentrations of the test compounds were determined by
venously administered at the initial dose (23.7 mg/kg), fol- HPLC measurement.
lowed by a constant-rate infusion (24.1 mg/kg/h) to reach the
Analytical Method Antipyrine, 4-OH, and 4-S were si-
target plasma concentration (100 mg/ml) through the cannula multaneously determined by the reversed-phase HPLC mea-
fastened to the left femoral vein throughout the experiment. surement according to our previous reports.12,14) Briefly, a
4-OH and 4-S (30 mg/kg and 100 mg/kg, respectively) were high performance liquid chromatograph (LC-10AD, Shi-
intravenously administered under a steady-state concentra- madzu, Kyoto, Japan) connected with an Inertsil ODS col-
tion of antipyrine at 1 h (for 4-OH and 4-S), 2 h (for 4-OH) umn (average particle size of 5 mm, 4.6 mm i.d.ϫ250 mm,
and 3 h (for 4-S) after the start of infusion, respectively.
GL Science, Inc., Tokyo) and an UV detector (SPD-10A,
Effect of 4-OH and 4-S on Tissue-to-Plasma Concentra- Shimadzu, Kyoto) operated at 254 nm were used. A mixture
tion Ratio (Kp) of Antipyrine: 4-OH and 4-S (30 mg/kg and of 50 mM phosphate buffer solution (pH 7.4) and methanol
100 mg/kg, respectively) were administered to the jugular (75 : 25, v/v) was used as the mobile phase. The flow rate was
vein at a steady-state of antipyrine (plasma concentration was 1.0 ml/min. Injection volumes were 30 ml, and the tempera-
100 mg/ml). Five minutes after such administration, blood ture was 40 °C. Five hundred microliters of methanol was
was collected from the abdominal aorta, and the rats were de- added to 100 ml of plasma sample to denaturalize the protein.
capitated immediately. Tissue samples of the kidney, brain, After precipitation of protein by centrifugation (3000 rpmϫ
liver, heart, spleen and lung were extirpated and homoge- 10 min), 60 ml of the supernatant filtered through a 0.45 mm
nized (15000ϫg) in isotonic phosphate buffer (pH 7.4) for 5 pore-size membrane filter (Japan Millipore Co., Yonezawa,
min. Five milliliters of ethyl acetate was added to 1 ml of the Japan) was injected into HPLC for measurement. Antipyrine,
homogenate and the mixture was shaken for 1 h. After the 4-OH and 4-S were separately detected as symmetrical
centrifugation (3000 rpm, 10 min and 4 °C), 4 ml of the upper peaks.
layer was collected and evaporated in vacuo. The residue was
dissolved in 300 ml of the mobile phase solvent for HPLC 4-OH, and 4-S after the intravenous administration were
measurement. Saline was used as a control. fitted to bi-exponential curve, CpϭA·exp(Ϫa·t)ϩ
Data Analysis Plasma concentrations (Cp) of antipyrine,
a
Effect of 4-OH and 4-S on Blood-Brain Barrier (BBB) B·exp(Ϫb·t), by the nonlinear least-squares regression pro-
Permeability of Antipyrine: 4-OH and 4-S were intra- gram17) with a weight of 1/Cp. Pharmacokinetic parameters
venously administered at the initial dose (4.40 mg/kg and were calculated from the estimated parameters, A, B, a and
6.13 mg/kg, respectively), followed by constant infusion b, where the area under the plasma concentration–time curve
(52.9 mg/kg/h and 44.2 mg/kg/h, respectively) to maintain (AUC ) was calculated from A/aϩB/b and the total body
the target plasma concentration (10 mg/ml and 50 mg/ml, re- clearance, CLtotal, was equal to Dose/AUC. The elimination
spectively) through the cannula fastened to the left femoral half-life, t1/2b, was calculated from ln 2/b. The mean resi-
vein throughout the experiment. Antipyrine (50 mg/kg) was dence time, MRT, was calculated as AUMC/AUC, AUMC
intravenously administered at a steady-state concentration of meaning the area under the first moment curve. The volume
4-OH (or 4-S) 120 min after the start of infusion. Five, 30, 60 of the central compartment, Vd1, was calculated as
or 120 min after the administration of antipyrine, the rats Dose/(AϩB). The apparent volume of distribution at steady-
were decapitated. Tissue sample of the brain was treated as state, Vdss, was calculated as Dose·AUMC/AUC 18,19) (Fig. 1).
described in ‘Effect of 4-OH and 4-S on Tissue-to-Plasma
Tissue-to-plasma concentration ratios (Kp) were estimated
Concentration Ratio (Kp) of Antipyrine’ and the resulting under the steady-state of antipyrine according to the follow-
sample was supplied for HPLC measurement. Saline was ing equations:
used as a control.
In Vitro Experiments Effect of 4-OH and 4-S on Pro-
KpϭCt/Cpϭ(Ct·VtϪCp·Ve)/Vi/Cp
tein Binding of Antipyrine: To 450 ml of rat plasma preincu-
bated at 37 °C for 2 min was added 50 ml of antipyrine in
saline (final concentration was 20 mg/ml) and the mixture
VeϭVt·IS
VtϭViϩVe
was incubated at 37 °C for 5 min. After incubation, the mix- where Ct and Cp are the concentrations of antipyrine in the
ture was immediately ultrafiltrated through ULTRAFREE tissue and in the plasma, respectively; Vt is the tissue volume;
C3LGC (Japan Millipore Co., Tokyo, Japan). Centrifugation Vi and Ve are the intracellular volume and the extracellular
was continued for 20—30 min at 5000ϫg until 10—15% of volume, respectively. The specific gravities of all tissues
the mixture was filtered. The filtrate was diluted with were assumed to be equal to 1. The Kp values of antipyrine
methanol (5-fold volume of the filtrate), and concentration of corrected by inulin space are shown in Fig. 5.
the free drug (unbound drug) was determined by HPLC mea-
surement.
To determine partition coefficient, the initial concentration
of antipyrine in the aqueous layer and its concentration in the
Effect of 4-OH and 4-S on Apparent Partition Coefficient aqueous layer after equilibrium were estimated, and finally
of Antipyrine: A mixture of 4 ml of n-octanol saturated phos- the concentration in the n-octanol layer after equilibrium was
phate buffer solution (50 mM, pH 7.4) containing 0.1 mM of determined. The equation is shown in
antipyrine and 0.1—1 mM of 4-OH (or 4-S) and 4 ml of n-oc-
PoctanolϭCoctanol/Cwaterϭ(CoϪCwater)/Cwater
tanol which was saturated with 50 mM phosphate buffer solu-
tion (pH 7.4) was severely shaken at room temperature for 30 where Coctanol is the concentration of the drug in the n-octanol
min. The mixture was then slowly shaken at 37 °C for 2 h. layer after equilibrium; Cwater is the concentration of the drug
The upper layer (n-octanol layer) was diluted with methanol in the aqueous layer after equilibrium; Co is the initial con-

May 2001
531
Fig. 1. Pharmacokinetic Model for Intravenous Administration
centration of the drug in the aqueous layer.
The permeability clearance of BBB (Kin) was determined
according to the graphical evaluation method of Blasberg et
al.20) and Gjedde.21) Usually the equation of mass balance is
shown by
dCbr/dtϭKin·CpϪKout·Cbr
(1)
where Cbr is the concentration in the brain, Cp is the concen-
tration in plasma, Kout is the transport clearance from the
brain to plasma. The efflux from the brain to plasma can be
neglected (CbrϽϽCp) immediately following administration of
the drug, so that Eqs. 2 and 3 can be derived from Eq. 1:
dCbr/dtϭKin·Cp
(2)
T
(3)
Cbr (T )ϭ Kin
Cpdt
∫
0
Therefore, Kin can be obtained using the concentration in the
brain at T (h) after administration of the drug (Cbr(T)) and
the area under the plasma concentration–time curve (AUC )
until T (h) after administration of the drug (͐0T Cpdt). The sin-
gle time point method22) using Eq. 3, however, sometimes af-
Fig. 2. Plasma Concentration–Time Curves of Antipyrine, 4-OH and 4-S
(50 mg/kg, i.v.)
Results are expressed as the meanϮS.E. (nϭ3—5).
fords an error in the revision of the dose of the remaining and 4-S on protein bindings of antipyrine, effects of 4-OH or
drug in the vascular. On the other hand, the graphical evalua- 4-S on apparent partition coefficient of antipyrine were per-
tion method20,21) using doses of the drug in the brain at sev- formed by the Student’s t-test and Tukey’s multiple compari-
eral T (h) after its administration (qtot(T)) is shown by son procedure (ANOVA). In all statistical testing, p values
less than 0.05 were considered to be statistically significant.
T
(4)
qtot(T )ϭ Kin
Cpdt ϩVdbr Cp (T )
∫
0
RESULTS
where Vdbr is the total value of plasma volume in the brain
and volume of extravascular compartment, Cp(T) is the con-
Pharmacokinetics of Antipyrine, 4-OH and 4-S The
centration in plasma at T (h) after administration of the drug. plasma concentration–time curves of antipyrine, 4-OH and 4-
Eq. 5 can be derived from Eq. 4:
S after intravenous administration (50 mg/kg, respectively)
are shown in Fig. 2. Pharmacokinetics parameters of these
compounds are summarized in Table 1. Compared with the
parent compound (antipyrine), 4-OH and 4-S were quite
T
(5)
qtot(T )/Cp(T )ϭ Kin
Cpdt /Cp (T )ϩVdbr
∫
0
From this equation, the regression line is obtained in which rapidly eliminated from plasma, in the order of 4-OH, 4-S,
slope and intercept are Kin and Vdbr, respectively. and antipyrine. The CLtotal values of 4-OH and 4-S were ap-
Statistical Analysis Statistical analyses of the pharma- proximately 20 times and 3.5 times as large as that of an-
cokinetic parameters, effects of 4-OH and 4-S on plasma tipyrine, respectively. Neither the administration of 4-OH nor
concentration–time curves of antipyrine, effects of bolus the 4-S production of antipyrine from 4-OH or 4-S was rec-
challenge with 4-OH and 4-S on plasma concentration pro- ognized in plasma.
file of antipyrine, effects of 4-OH and 4-S on tissue-to-
Effect of 4-OH and 4-S on Plasma Concentration–Time
plasma concentration ratio (Kp) of antipyrine, effects of 4-OH Curves of Antipyrine Plasma elimination profiles of intra-

532
Vol. 24, No. 5
Table 1. The Pharmacokinetic Parameters of Antipyrine, 4-OH and 4-S
Parameters
Antipyrine
4-OH
4-S
A (mg/ml)
B (mg/ml)
148Ϯ46
82.4Ϯ17.4
18.1Ϯ1.3
0.39Ϯ0.12
10.6Ϯ1.4
6.8Ϯ0.01
1.02Ϯ0.23
235Ϯ65
73.4Ϯ0.3
40.8Ϯ1.1
23.9Ϯ0.6
6.3Ϯ0.04
5.7Ϯ0.1
12.6Ϯ0.3
12.0Ϯ0.03
438Ϯ3
281Ϯ4
130Ϯ2
a (hϪ1
)
22.1Ϯ1.2
2.96Ϯ0.03
8.8Ϯ0.8
9.0Ϯ0.2
7.2Ϯ0.1
122Ϯ1
b (hϪ1
)
k12 (hϪ1
k21 (hϪ1
k10 (hϪ1
)
)
)
Vd1 (ml/kg)
Vdss (ml/kg)
CLtotal (ml/h/kg)
610Ϯ209
254Ϯ119
635Ϯ13
5285Ϯ49
242Ϯ16
883Ϯ11
The dose of each drug was 50 mg/kg. Results are expressed as the meanϮS.E.
(nϭ3—5).
Fig. 4. Effect of Bolus Challenge with 4-OH and 4-S on Plasma Concen-
tration Profile of Antipyrine
pϽ0.05, pϽ0.01; significantly different from plasma concentration before bolus
challenge with 4-OH and 4-S. Results are expressed as the meanϮS.E. (nϭ4).
Fig. 3. Effects of 4-OH and 4-S on Plasma Concentration–Time Curves of
Antipyrine (50 mg/kg, i.v.)
pϽ0.05,
pϽ0.01; significantly different from control (saline). Results are ex-
pressed as the meanϮS.E. (nϭ3—5).
venously administered antipyrine (50 mg/kg) under the
steady-state concentration of 4-OH or 4-S are shown in Fig.
3. Concentrations of 4-OH and 4-S in plasma were kept at
constant levels of about 10 mg/ml and 50 mg/ml, respectively.
Plasma elimination of antipyrine was significantly deceler-
ated by 4-OH, but was not affected by 4-S.
Effect of Bolus Challenge with 4-OH and 4-S on
Plasma Concentration Profile of Antipyrine The effect
of intravenously administered 4-OH (30 mg/kg) and 4-S (100
mg/kg) on steady-state plasma concentration of antipyrine is
shown in Fig. 4. The plasma concentration of antipyrine was
drastically decreased by the intravenous coadministration of
4-OH or 4-S. However, this phenomenon was transient and
the plasma concentration of antipyrine was restored to the
original level within 10—40 min.
Fig. 5. Effect of 4-OH and 4-S on Tissue-to-Plasma Concentration Ratio
(Kp) of Antipyrine
pϽ0.05; Significantly different from control (saline). Results are expressed as the
meanϮS.E. (nϭ4).
Effect of 4-OH and 4-S on Tissue-to-Plasma Concen-
tration Ratio (Kp) of Antipyrine The Kp values of an-
tipyrine 5 min after the intravenous bolus administration of 4-
OH or 4-S under the steady-state concentration of antipyrine
are shown in Fig. 5. These Kp values were increased signifi-
cantly in the brain and heart by the administration of 4-OH,
but were not affected by 4-S.
Effect of 4-OH and 4-S on Protein Binding of An-
tipyrine As shown in Fig. 6, approximately 15% of an-
tipyrine was bound to plasma protein. This was reduced with
the addition of 4-OH.
cient of Antipyrine Apparent partition coefficient of an-
tipyrine in the presence of 4-OH (or 4-S) is shown in Fig. 7.
This partition coefficient increased with the addition of 4-OH
until three-fold 4-OH was added, however, it decreased with
the addition of more than three-fold 4-OH. In contrast, the
partition coefficient of antipyrine decreased with the addition
of only one-fourth-fold 4-S, and no further change in this
value was observed by a rise in this concentration.
Effects of 4-OH and 4-S on Blood-Brain Barrier (BBB)
Permeability of Antipyrine The concentration of an-
tipyrine in the brain after its intravenous administration under
the steady-state plasma concentration of 4-OH or 4-S was
Effects of 4-OH and 4-S on Apparent Partition Coeffi-

May 2001
533
Table 2. The Permeability Clearance of BBB (Kin) and The Total Distribu-
tion Volume (Vdbr) in Brain Determined by The Graphical Evaluation Method
Kin
(ml/min/g)
Vdbr
(ml/g)
Condition
Control
ϩ4-OH
ϩ4-S
29.1
137
7.56
226
179
129
Graphical evaluation method using doses of the drug in the brain at several T (h)
after administration of the drug (qtot(T)), shown in qtot(T)ϭKin ͐0T CpdtϩVdbr·Cp(T) (Eq.
1), where Kin is the permeability clearance of BBB, ͐0T Cpdt is the area under the
plasma concentration–time curve until T (h) after the administration, Vdbr is the total
value of plasma volume in the brain and volume of the extravascular compartment,
Cp(T) is the concentration in plasma at T (h) after the drug administration. Equation 2
can be derived from the Eq. 1: qtot(T)/Cp(T)ϭKin ͐
0T Cpdt/Cp(T)ϩVdbr(Eq. 2). From this
equation, the regression line is obtained in which slope and intercept are Kin and Vdbr
,
respectively (see Fig. 8).
Fig. 6. Effect of 4-OH and 4-S on Protein Bindings of Antipyrine
pϽ0.05, pϽ0.01; Significantly different from bound ratio without 4-OH and 4-
S. Results are expressed as the meanϮS.E. (nϭ3).
Kin of antipyrine under the steady-state plasma concentration
of 4-S was 7.56 ml/min/g, which was about one-fourth that of
the control. Vdbr was decreased by the concomitant adminis-
tration of either 4-OH or 4-S.
DISCUSSION
We have already demonstrated that the pharmacokinetics
of drugs are sometimes influenced by the biopharmaceutical
interactions with their metabolites.5—10,12—14) In this report,
the effects of the metabolite of antipyrine (4-OH) and its sul-
fate (4-S) on the pharmacokinetics of antipyrine which was
the centrally acting drug were investigated in rats.
4-OH under a steady-state concentration significantly de-
celerated the plasma elimination profile of intravenously ad-
ministered antipyrine, while 4-S did not affect it. (see Fig. 3).
Similarly, the Kp value of antipyrine increased in the brain
and heart by the concomitant use of 4-OH but was not af-
fected by 4-S (see Fig. 5). It is therefore possible that 4-OH
inhibited the metabolism of antipyrine, thus increasing its
plasma concentration and enhancing its transition to tissues
as a result. The plasma concentration of antipyrine was dras-
tically decreased by the intravenous bolus coadministration
of 4-OH (or 4-S) at steady-state plasma concentration of an-
tipyrine, and this phenomenon was stronger when 4-OH was
used than 4-S (see Fig. 4). It seems that this decrease was
caused by the transition of antipyrine to tissues.
Fig. 7. Effect of 4-OH or 4-S on Apparent Partition Coefficient of An-
tipyrine
pϽ0.05,
pϽ0.01; Significantly different from apparent partition coefficient of
antipyrine without 4-OH and 4-S. Results are expressed as the meanϮS.E. (nϭ3).
In general, an intravenously administered drug must pass
through the plasma membrane for distribution to the tissues,
and one of the factors allowing membrane transport are the
protein binding ratio and partition coefficient of the drug.
The protein binding ratio of antipyrine was reduced by the
concomitant use of 4-OH (see Fig. 6) in this study. But, con-
sidering the distribution volume of antipyrine (235Ϯ65 ml/kg
for Vd1 and 610Ϯ209 ml/kg for Vdss, see Table 1), the increase
of unbound antipyrine in plasma by 4-OH seemed to make a
relatively small contribution to the enhancement of the tran-
sition to tissues.
Fig. 8. Effect of 4-OH and 4-S on Blood-Brain Barrier (BBB) Permeabil-
ity of Antipyrine
The partition coefficient of antipyrine was apparently in-
creased by the concomitant use of 4-OH (see Fig. 7). One of
the effects changing the partition coefficient of the compound
is the salting-out effect. At first, we speculated that this effect
caused the increase of the partition coefficient of antipyrine,
but 50 mM phosphate buffer solution was employed in this
experiment, and the concentration of antipyrine and 4-OH
determined and plotted according to the graphical evaluation
method.20,21) The result is shown in Fig. 8. The permeability
clearance of BBB (Kin) and the total distribution volume
(Vdbr) are summarized in Table 2. Kin of antipyrine under the
steady-state plasma concentration of 4-OH was 137 ml/min/g,
which was about 5 times as large as that of the control. But

534
Vol. 24, No. 5
ified with polyamine24) have been suggested as the drug de-
livery system to the brain. Increase of BBB permeability by
stress exposure has also been reported.25) From another view-
point, increase of drug transition to the brain by the concomi-
tant use of 4-OH as recognized in this study is information
useful for application of a drug combination as a biodistribu-
tion promoter.
were only 0.10 mM and 0.025—1.0 mM, respectively. That is,
the salt concentration of the buffer solution was 50—2000-
fold those of antipyrine and 4-OH. It would seem very diffi-
cult for the migration of a small amount of 4-OH from the
water layer to the organic layer to have this effect on the par-
tition coefficient of antipyrine under our conditions. This fac-
tor of the salting-out effect was thus neglected, and at present
it is not clear why the partition coefficient of antipyrine was
increased with the concomitant use of 4-OH. Furthermore, if
the change in this coefficient were to cause an enhancement
in its Kp value, it seems that Kp values would increase in all
organs; this was not the result, however. We therefore believe
that there was some kind of interaction between antipyrine
and 4-OH, and that this interaction increased the partition co-
efficient of antipyrine and also specifically caused the en-
hancement of its Kp value in the brain and heart. At present,
however, we have no plausible explanation of what kind of
interaction this was or why this phenomenon occurred.
The concentration of antipyrine in the brain after its intra-
venous administration at a steady-state plasma concentration
of 4-OH or 4-S was determined (see Fig. 8), and the Kin
value at the concentration of 4-OH was about 5 times as
large as that of the control. Vdbr value was decreased by the
concomitant administration of either 4-OH or 4-S (see Table
2). From these results, it seems that the enhancement of the
BBB permeability of antipyrine caused by 4-OH resulted in
the increase of its Kp value in the brain.
Acknowledgment We are grateful to The SC-NMR Lab-
oratory of Okayama University for 200 MHz 1H-NMR exper-
iments.
REFERENCES
1) Niwa H., Nakayama T., Arch. Pract. Pharm., 30, 206—211 (1970).
2) Thomas B. H., Zeitz W., Coldwell B. B., J. Pharm. Sci., 63, 1367—
1370 (1974).
3) Price V. F., Gale G. R., Res. Commun. Chem. Pathol. Pharmacol., 57,
249—260 (1987).
4) Tsuji A., Sato H., Okazaki E., Nagata O., Kato H., Biochem. Pharma-
col., 37, 4408—4411 (1988).
5) Niwa H., Nakayama T., Yakugaku Zasshi, 88, 535—541 (1968).
6) Niwa H., Nakayama T., Yakugaku Zasshi, 88, 542—548 (1968).
7) Niwa H., Nakayama T., Yakugaku Zasshi, 88, 843—846 (1968).
8) Nakayama T., Yakugaku Zasshi, 88, 847—851 (1968).
9) Niwa H., Nakayama T., Bull. Tohoku Pharmaceutical Collage, 16, 1—
13 (1969).
10) Niwa H., Nakayama T., Bull. Tohoku Pharmaceutical Collage, 16,
15—23 (1969).
11) Studenberg S. D., Brouwer, K. L. R., Pharmacok. Biopharm., 21,
175—194 (1993).
Further study on the mechanism of the distribution promo-
tion effect will be performed in the near future.
12) Nakayama T., Sawamoto T., Kurosaki Y., Kimura T., Int. J. Pharma-
ceut., 121, 55—63 (1995).
13) Sawamoto T., Kurosaki Y., Kimura T., Nakayama T., Biol. Pharm.
Bull., 19, 77—82 (1996).
CONCLUSION
14) Sawamoto T., Kurosaki Y., Sasaki K., Kimura T., Nakayama T., Int. J.
Pharmaceut., 146, 181—191 (1997).
The Kp values of antipyrine increased significantly in the
brain and heart with the concomitant administration of 4-OH
under the steady-state concentration of antipyrine in plasma,
but were not affected by 4-S. On the other hand, the partition
coefficient of antipyrine was apparently increased by the con-
comitant use of 4-OH. It seemed this phenomenon would be
concerned with a mechanism to increase the Kp values of an-
tipyrine in the brain and heart. However, it was not clear what
kind of interaction between antipyrine and 4-OH acted to en-
hance the partition coefficient of antipyrine. The protein
binding ratio of antipyrine seems to make relatively small
contribution to the enhancement of its transition to tissues.
The enhancement of the BBB permeability of antipyrine
caused by 4-OH was believed to be concerned with increase
15) Nakayama T., Sawamoto T., Karino T., Matsumura M., Sasaki K.,
Kurosaki Y., Kimura T., Biol. Pharm. Bull., 20, 522—529 (1997).
16) Hukki J., Myry J., Acta Chem. Scand., 13, 174—177 (1959).
17) Yamaoka K., Tanigawara Y., Nakagawa T., Uno T., J. Pharmacobio-
Dyn., 4, 879—885 (1981).
18) Etagere M., Nakamura H., Maruyama T., Imamura Y., Takadate Y.,
Chem. Pharm. Bull., 37, 498—501 (1989).
19) Yamaoka K., Nakagawa T., Uno T., J. Pharmacokinet. Biopharm., 6,
547—558 (1978).
20) Blasberg R. G., Fenstermacher J. D., Patlak C. S., J. Cereb. Blood Flow
Metab., 3, 8—32(1983).
21) Gjedde A., J. Neurochem., 36, 1463—1471 (1981).
22) Ohno K., Pettigrew K. D., Rapoport S. I., Am. J. Physiol., 235, 299
(1978).
23) Traci L. M., David E. B., Alvin V. T., Jr., Warren J. B., Chris R. M.
David D. A., Neurosci. Let., 258, 25—28 (1998).
of the Kp value of antipyrine in the brain. Recently various 24) Joseph F. P., Geoffry L. C. Jagjit S. G., J. Neurochem., 71, 1651—1660
(1998).
methods such as the BBB basic amine transporter, which is
25) Ivana S., Dmitrij T., Daniela J., Brain Res. Bull., 45, 175—178 (1998).
used for the transition of hydrophilic and charged com-
pounds to the brain,23) and nerve growth factor which is mod-