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Antipyrine estimations in the rabbit using gas-liquid chromatography: A reliable method for studying factors affecting oxidative drug metabolism
Antipyrine Estimations in the Rabbit Using Gas-liquid Chromatography: A Reliable Method for Studying Factors Affecting Oxidative Drug Metabolism
D.M.
CHAMBERS AND G.C.
The application of antipyrine is described
JEFFERSON
of a gas-liquid in plasma
chromatographic
for the estimation
and is compared
(GLC) method
of antipyrine
with previous
to measurements
half-life
GLC methods.
(T,,J
in the rabbit
It has been established
that the estimation of antipyrine can be achieved with acceptable precision and that rabbits can act as their own controls in the study of factors that might alter T112. lproniazid phosphate, 50 mg/kg IP, was shown to produce a mean increase of 170% in Tli2. Key Words:
Antipyrine;
Biotransformation;
Iproniazid;
Rabbit
INTRODUCTION Antipyrine half-life (T,,J estimations have been widely used to assess the effect of factors that influence hepatic oxidative drug-metabolizing enzymes. These factors include drugs that cause inhibition or induction of these enzymes and a number of diseases (Stevenson, 1977). Whilst T,,2 has been measured extensively in man, animal studies have also made an important contribution to this field. Measurements of T,,2 have been undertaken in a variety of animal species including mouse (Quinn et al., 1958; Chambers and Jefferson, 1977), rat (Quinn et al., 1958; Sotaniemi, 1973; Aarbakke, 19781, dog (Vesell et al., 1973; Kampffmeyer, 19741, rhesus monkey (Branch et al., 19741, baboon (Down, 1976), horse (Powis and Snow, 19781, and even the quokka, a marsupial (McManus and Ilett, 1979). In general the antipyrine is estimated from blood samples although Welch et al. (1975) carried out estimations using rat saliva. For the smaller species, mouse and rat, T,,* estimates often rely on data from pooled blood samples whereas the larger species, by sequential blood sampling, allow a T 112 estimate from each animal. However, T,,2 has been estimated from individual rats, Bakke et al. (1974). It is surprising that there have been few measurements of T7,2 in vivo in the rabbit (Quinn et al., 1958; Statland et al., 1973; Van Peer et al., 1978; McManus and Ilett, 1979) as sequential blood sampling is eminently suitable in this species and because each animal might be able to act as its own control in studies of factors likely to change T,,*. Statland et al. (1973) and Van Peer et al. (1978) used rabbits as their own From the Pharmacology Section, Department of Pharmacy, Heriot-Watt University, 79 Grassmarket, Edinburgh, Scotland. Address reprint requests to D.M. Chambers, Pharmacology Section, Department of Pharmacy, Heriot-Watt University, 79 Grassmarket, Edinburgh EHI 2HJ, Scotland. Received March 1980; revised and accepted May 1980. 59 Journalof
Pharmacological
0 1981 Elsevier
North
Methods
Holland,
5, 59-66 f1981)
Inc., 52 Vanderbilt
Avenue, New York, NY 10017
OlMJ-5402/81/0059008/$02.50
60
D.M. Chambers and G.C. Jefferson controls method.
although their data gave little information about the precision of the Furthermore, their results did not show any significant change in T,/2 after
exposure before
of the animals
to certain
metabolic
studies,
monstrable
further
lengthening
was required.
Information
Therefore,
Most
workers
on the precision
of T 1,2 in the presence
It is known
that in the rabbit
antipyrine is almost exclusively zymes prior to elimination. Brodie
pretreatments.
the use of TT,* in the rabbit can be recommended
have estimated
antipyrine
et al. (1949) or the gas-liquid
et al. (1973) or Lindgren
chromatographic
tographic
(Shargel
and
of an established
and the de-
enzyme
et al., 1968),
by oxidative
Occasionally
inhibitor
as in man,
drug-metabolizing
by the spectrophotometric
chromatographic
et al. (1974).
thin-layer
that
of the method
(Yoshimura
metabolized
we considered
for drug interaction
method
(GLC) methods
radiometric
(Bakke
enof
of Prescott et al., 1974),
(Welch et al., 1975) and high-pressure liquid chromaet al., 1979) methods have been used. The present work uses a
GLC method based on that previously reported (Chambers and Jefferson, 1977) and which in turn was modified from that of Prescott et al. (1973). By contrast all of the previous metric
antipyrine method
T,,* determinations
except McManus
in the rabbit
and llett
have used the spectrophoto-
(1979) who used the radiometric
method.
METHODS Male New Zealand on commercial
white
rabbits
rabbit pellets
weighing
(McGregor)
rabbits were started at 09 hours 00 minutes at 21 *
between
3 and 4 kg were maintained
and water ad libitum.
All experiments
with the ambient temperature
on
maintained
1°C.
Antipyrine
T,,, Estimations in the Rabbit
In each rabbit antipyrine Tqr2 was estimated interval of between 21 and 28 days. Antipyrine, jected into a marginal taken for analysis
ear vein followed
from
the marginal
on two occasions separated by an 50 mgikg in sterile water, was in-
by 1000 IU of heparin.
ear vein contralateral
40, 60, 80, 100, 120, and 140 minutes.
Blood
loss
Blood samples
to the injected
were
ear at 20,
can cause a reduction
in drug
metabolism (Cumming et al., 1971) and in the present work, the total loss was 5 ml/ kg, a volume thought unlikely to have caused a reduction in antipyrine metabolism. A subsequent
study
on metabolic
inhibition
was performed in which three rabbits (control) followed T ,,2 estimation
received pretreatment with saline prior to the first by iproniazid phosphate (50 mg/kg) pretreatment All pretreatments
were given intraperitoneally
Analysis of Antipyrine To 1 ml samples (Oxford taining
Dispensor)
to the second prior
estimation.
to antipyrine.
in Plasma
of plasma
(Oxford
and 1 ml chloroform
12.5 kg phenacetin
screw-capped
prior
one hour
(Oxford
glass centrifuge
Sampler)
were added 0.2 ml of 5M NaOH
(McFarlan
Pipettor)
Smith,
as an internal
tubes with Teflon
liners
anesthetic reference
grade) constandard
in
(Sovirel).
These tubes were placed horizontally in a metabolic shaker and oscillated wise for 15 minutes at a rate of 65 per minute. The tubes were centrifuged
lengthat 3000
Antipyrine Half-life in the Rabbit g for 10 minutes. pipette
The
into individual
organic
phase was removed
tapered glass centrifuge
ness by placing the tubes
from
tubes,
each tube with
a Pasteur
and then evaporated
in a water bath at 90°C for 10 minutes.
The
to dry-
residues
were
redissolved in 20 PI of chloroform using a vortex mixer and kept on ice. Three j-4 aliquots of this solution were injected onto a 2 m x 1.75 mm o.d. glass column packed with 100-120
mesh Gas Chrom
20M. The temperature
of the column
and flame ionization
detector
rate of 90 ml/minute.
Antipyrine
antipyrine:
phenacetin
with
We
established
0.15-7.75)
that a plot
by the equation error
concentrations
gas was nitrogen
were calculated using integral
to corresponding
of peak integral concentration
y = 0.0857x-0.0265 of the regression
ratios
(2-90
ratios from
ratios
standards
pre-
of 30 pg/ml.
antipyrine:phenacetin
j.r.g/ml) was a straight
with an error coefficient,
port
used at a flow
to rabbit plasma to give a concentration
against antipyrine
standard
was 250°C. The carrier reference
pared by adding antipyrine
Q coated with 0.5% SE 30 and 0.5% carbowax was 220°C and that of both the injection
(range
line described
(S&I/~%) of 0.27 where
Sb is the
b, of the line.
Materials For injection, (Roche)
in sterile
antipyrine
(BDH
saline
(Polyfusor,
Chemicals) Boots)
in sterile
water, or iproniazid
were prepared
immediately
phosphate before
use.
RESULTS Estimations of Antipyrine Antipyrine regression
T,,? in Rabbits on Two Occasions
Tli2 was determined
for each rabbit from the slope of the least squares
line fitted to the plot of log plasma antipyrine
concentration
against time.
I 20
40
60
Time
80
100
I20
140
(min)
FIGURE 1. Antipyrine half-life in rabbit 2 (Table 3) after pretreatment with saline (0) (control) or iproniazid phosphate (O), 50 mg/kg IP. Pretreatments given one hour before antipyrine, 50 mg/kg IV. Interval between half-life determinations was 21 days.
61
62
D.M. Chambers and C.C. Jefferson TABLE 1
Antipyrine
Pharmacokinetics
in the Rabbit APPARENT
HALF-LIFE
(T,,,)
(min)
Mean SE Statistical difference between estimates (Pairedt test)
OF
(v,)
PLASMA
CLEARANCE
(PC)
(ml.min-'.kg ')
(Ikg~')
ESTIMATE? RAEEIT
VOLUME
DISTRIBUTION
ESTIMATESa
ESTIMATES”
1st
2nd
1st
2nd
1st
2nd
49.8 48.7 42.5 46.1 79.0 88.3 97.4 53.9
45.0 52.4 52.3 49.5 83.5 91.0 82.0 72.9
0.821 0.739 1.113 0.699 0.821 0.774 0.799 0.669
0.833 0.819 0.866 0.890 0.762 0.892 0.909 0.846
11.43 10.53 18.15 10.52 7.20 6.07 5.68 8.60
12.83 10.83 11.47 12.45 6.32 6.79 7.68 8.05
63.2 7.6
66.1 6.4
0.804 0.048
0.852 0.017
9.77 1.42
9.55 0.93
>0.05
>0.05
10.05
a Intervalbetween 1st and 2nd estimateswas 21-28 days.
Table 1 shows the estimates of T,,* on two occasions in each of eight rabbits. corresponding apparent volumes of distribution (V,) and plasma clearances are also given. PC was calculated (Riggs, 1963) from the equation: pC = Dose antipyrine
(mg/kg)
Co
The (PC)
.-0.693 TII2
where Dose antipyrine
=
VD
CO
and Co is the theoretical plasma antipyrine concentration at zero time. There was no significant change in T,,*, V,, or PC in the eight rabbits between estimations (p > 0.05 by paired t-test). Analysis of variance on T7,* values (Table 2) showed a significant inter-animal variation (p < 0.001).
TABLE 2 Table 1
Analysis of Variance of Antipyrine
DEGREES SOURCE
OF VARIATION
Between rabbits Within rabbits Total
SUM
OF SQUARES
5214.97 385.01 5599.97
f = 15.48 (d.f.= 7,8);p < 0.001.
FREEDOM
7 8 15
r,,, Results from
OF MEAN
SQUARE
745.00 48.13
Antipyrine Half-life in the Rabbit TABLE 3
Effect of lproniazid
Phosphate 50 mg/kg IP on Antipyrine Pharmacokinetics APPARENT
HALF-LIFE
(T,,z)
(min)
___~ 1 2 3
OF
(vo)
PLASMA
CLEARANCE
(ml.min-
(Ikg-‘1 ESTIMATE?
ESTIMATESd RABBIT
VOLUME
I~ISTRIBUTION
(PC)
‘.kg- ‘1
ESTIMATES=
1st ____.~ 100.9 81.2 81.6
2nd
1st
2nd
1st
2nd
199.4 143.3 168.2
0.678 0.732 0.662
0.636 0.697 0.432
4.72 6.25 5.62
2.21 3.37 1.78
87.9 6.5
170.3 16.2
0.691 0.021
0.588 0.080
5.53 0.44
2.45 0.47
Mean SE Statistical differences between estimates (Paired t test)
>0.05
CO.05
CO.05
a Interval between 1st and 2nd estimates was 21-28 days.
Effects of lproniazid
on Antipyrine
Pharmacokinetics
The effect of iproniazid phosphate 50 mg/kg IP in three rabbits is shown in Table 3. There was a significant lengthening of T,,2 and a corresponding reduction in PC. Figure 1 shows the change in T1,2 after iproniazid in one of the rabbits. The percentage increase of T1,2 in the three rabbits after iproniazid ranged from t-76.6% to +106.1% (mean +93.4%). The error of each regression line from which T,,2 was calculated was determined. The mean error (S&b%) ? SE from all the estimates shown in Tables 1 and 3 was 6.3 + 0.58% (n = 22). DISCUSSION GLC methods are widely used for the analysis of antipyrine in body fluids and, compared with the spectrophotometric method of Brodie et al. (1949), they have the advantage that the presence of metabolites does not contribute to antipyrine estimations (Vesell and Passananti, 1973). The experimental conditions under which the GLC methods are used have varied widely. In our work the estimation of antipyrine concentrations from integral ratios antipyrine:phenacetin represents a departure from the use of peak height ratios by previous workers (Prescott et al., 1973; Huffman et al., 1974; Chambers and Jefferson, 1977). The relationship between our peak integral ratios and antipyrine concentration is described by a straight line with an error of 0.27%. Details of the precision of such standard curves has not been given in previous work using peak heights. The method we adopted for shaking during the antipyrine extraction avoids the problem of emulsion formation at the water:chloroform interface that was reported by Prescott et al. (1973) and that we had previously seen (Chambers and Jefferson, 1977). This had caused occasional difficulties in analysis. Huffman et al. (1974)
63
64
D.M. Chambers and C.C. Jefferson claimed from
that erratic
a double
single
results
were obtained
extraction
extraction
with
here only
if emulsions
chloroform
were formed.
(Chambers
caused a 3% reduction
The
and Jefferson,
in extraction
change
1977) to the
efficiency
with
no
loss of precision. From 22 antipyrine TIr2 estimates a mean extraction efficiency of 88 t 1.6% SE was achieved. The extraction efficiencies were calculated from a comparison
of the peaks obtained from extracted antipyrine
plasma) with
peaks obtained
phenacetin preferable
in chloroform. antipyrine
(1973).
latter
The
The
recovery
solution
procedure
solution
a known
measure
of antipyrine
extracted fails
to measure
deviation
from
kg with
an error
allowing
with the 5 kg/ml a sensitivity viation
of 9.3%
detection
of Prescott
to 0.5 kg/ml
of the Lindgren
antipyrine
the
using
or within
loss of antipyrine
from
work
of this
assess
of the species
the method.
differences
known
IP. This
compares work
the first
treatment
well with
(Table
I),
with
the values
values
in seven
no evidence
imme-
was no measurable of antipyrine
has not yielded
and
sufficient the rabbit
of certain compounds given antipyrine
of 63.2 and 66.1 minutes
that show
et al., 1976)
et al. (1958) included rabbits
de-
Subsequent
for antipyrine
amounts
by others,
Quinn
kg/ml.
There
that
concentration
(Van Boxtel
analysed
in the metabolism
a mean T,,2 of 63 minutes
present
method
at -20°C.
rabbit plasma containing
compared
as the standard
in the antipyrine
were either
days after storage
stored at -20°C over a period of 82 days. Work on T,,* in the rabbit previously reported to fully
of 0.5 kg/ml
in the range 10.1-15.1
a modification
fourteen
concentration
from the rabbit suggest
especially
method was as high as 10.1%
produced even larger errors. Plasma samples in the present
in a study
by either
et al. (1974) was of increased
concentrations
is not required,
pg/ml and only fell to 3.9%
determined
retained
to
et al.
4.4%, at the 5 kg/ml concenof 4.4% for the concentration
et al. (1973). Our analyses
group
data on which
plasma given relative
over the antipyrine
of Lindgren
of antipyrine
range 0.5-5.0
diately
was
at 5 kg/ml.
work
by this
and
extraction
as used by Prescott
antipyrine
for our method
claim made for the CLC method
sensitivity,
of antipyrine
of antipyrine
or by the plasma.
mean standard
The
(30 t_r.g/mlin rabbit
solution
simultaneously
range S-60 kg/ml was 3.4% with the greatest error, tration. Prescott et al. (1973) reported a mean error range S-50
standards
unextracted
It was felt that this
to the percentage
an aqueous aqueous
from
and
50 mg/kg
determined
in the
for enzyme-induction
after
antipyrine.
Repeated measurements of antipyrine TI,2 were made by Statland et al. (1973) in each of six rabbits after antipyrine doses of 50 mg/kg IM on days 1, 8, and 14. The results show little change in the mean TIlz values on the three occasions, being 60, 65.4 and 65.4 minutes respectively. If the percentage changes in TT12from first to second and first to third determinations are calculated for each rabbit from these results, it is found that they range from -23% to +57% (mean +15.0%) and from -25%
to +76%
(mean
+18.7%),
respectively.
In the present
work
the correspond-
ing changes from Table 1 range from - 15.8 to + 35.1% (mean + 7.1%). was given by Statland of the precision of individual T,,* determinations Furthermore
their
regression
lines
of log antipyrine
concentrations
No estimate et al. (1973). on time were
Antipyrine
Half-Life in the Rabbit
determined subjectively. It was noted that they only took blood samples at 80, 160, and 240 minutes after antipyrine injections compared with the seven samples taken over 140 minutes in experiments reported here. Van Peer et al. (1978) showed a change in mean T,,2 in five rabbits given saline from 73.8 to 72 minutes over a period of five days. These T1,2values again compare well with those quoted above. As no individual T,,2 values were given by Van Peer et al. (1978) and as there is no information on the errors of their TI12regression lines it is again difficult to gain information on the precision of the method. Our mean error of 6.3% for the regression lines of log antipyrine concentration on time suggests that our analytical performance is sound (Robinson, 1971) and compares well with a value of 7.9% reported from work in man (Vestal et al., 1975). The present T,,2 results indicate that intra-animal variation is less than inter-animal variation (Table 2), a comparable finding in respect of antipyrine T,,2 in man has been reported by Lindgren et al. (1974). As well as antipyrine Tl12 values, we have calculated the corresponding PC values in the rabbit. Smith and Rawlins (1974) discussed the use of PC measurements as an alternative to T,,2 for estimating drug metabolism. They claim that this use of T ,,2 was valid only if it could be assumed that the V, was constant in different individuals. We have, however, used each rabbit as its own control and shown that V, in a given rabbit had not altered between T,,, estimations (Table 1). Furthermore, a correlation was established in the rabbit between plasma antipyrine T 1,2 and liver microsomal antipyrine hydroxylase activity by Statland et al. (19731, who suggested that T ,,* measurements serve as a reliable index of antipyrine hepatic metabolism rather than its disposition or renal excretion. These findings, together with evidence of a lack of enzyme induction in the present work, indicate that with a suitable interval between determinations rabbits can act as their own controls in the investigation of potential factors causing variation in antipyrine Tl,*. Potential factors affecting T,,, investigated in the rabbit included carbon monoxide (Statland et al., 1973) and chemically-induced renal failure (Van Peer et al., 1978), both of which were without effect on T,,2, but there have been no reported investigations in the presence of drugs. We have shown in the rabbit that iproniazid, a compound that is known to inhibit hepatic mixed function drug-metabolizing enzymes (Laroche and Brodie, 1960), produced a marked lengthening of T,,* with a corresponding reduction in its PC and our results suggest that the use of antipyrine Tl,2 determinations in the rabbit may be of value in studies on oxidative drug metabolism. We gratefully acknowledge
the gift of iproniazid phosphate from Roche.
REFERENCES Aarbakke J (1978) Disposition and oxidative metabolism of antipyrine in the rat. Acta Pharmacol et Toxic0143
Bakke, OM,
:64-68. Bending
M, Aarbakke
J, Davies DS
(1974) ‘%Antipyrine as a model compound in the study of drug oxidation and enzyme induction in individual surviving rats. Acta Pharmacol et Toxicol35:91-97.
65
66
D.M. Chambers and G.C. Jefferson Branch RA, Shand DG, Wilkinson GR, Nies AS (1974) Increased clearance of antipyrine and dpropranoloi after phenobarbital treatment in the monkey. 1. Clin fnvest 53:1lOl-1107. Brodie BB, Axelrod I, Soberman R, Levy BB (1949) The estimation of antipyrine in biological materials. /. Bio/ Chem 179:25-29. Chambers DM, Jefferson GC (1977) Some observations on the mechanism of benzodiazepine-barbiturate interactions in the mouse. f?r j Pba~maco/ 60:393-399. Gumming, JF, McClung HW, Mannering G] (1971) The effect of hemorrhage on the rate of hexobarbital biotransformation in the dog. / Pharmacol Exp Ther 178:595-601. Down WH (1976) Species differences in drug metabolism; in vivo parameters of hepatic drugmetabolising enzyme activity in the baboon. In Microsomes and Drug Oxidation. Ed., V Ullrich. 0xford:Pergamon Press, pp. 516-519. Huffman DH, Shoeman DW, Azarnoff DL (1974) Correlation of the plasma elimination of antipyrine and the appearance of 4-hydroxyantipyrine in the urine of man. Biochem Pharmaco~ 23:197-201. Kampffmeyer HG (1974) Metabolic rate of phenacetin and of paracetamol in dogs before and after treatment with phenobarbital or SKF 525 A. Biochem Pharmacol23: 713-724. Laroche M, Brodie BB (2960) Lack of relationship between inhibition of monoamine oxidase and potentiation of hexobarbital hypnosis. f Pharmacol Exp Ther 130:134-137. Lindgren S, Collste P, Norlander B, Sjoqvist F (1974) Gas chromatographic assessment of the reproducibility of phenazone plasma half-life in young health volunteers. Eur/ C/in Pharmaco/7:381-385. McManus ME, llett KF (1979) Comparison of rate of hepatic metabolism in vitro and half-life for antipyrine in vivo in three species. Xenobiotica 9:107-118. Powis G, Snow DH (1978) The effects of exercise and adrenaline infusion upon the blood levels of propranolol and antipyrine in the horse. / Pbarmaco/ Exp 7’her 205:725-731, Prescott LF, Adjepon-Yamoah KK, Roberts E (1973) Rapid gas-liquid chromatographic estimation of antipyrine in plasma. / Pharm Pharmacol 25:205-207. Quinn GP, Axelrod J, Brodie BB (1958) Species, strain and sex differences in metabolism of hex-
obarbitone, amidopyrine, antipyrine and aniline. Biochem Pharmacol1:152-159. Riggs DS (1963) 7’he ~afhema~ica/ Approach to fhys;o/ogicai Problems. Baltimore: Williams and Wilkins, p. 127. Robinson R (1971) Clinical Chemistry mation. London: Griffin, pp. 1-18.
and Auto-
Shargel L, Cheung W, Yu ABC (1979) High-pressure liquid chromatographic analysis of antipyrine in small plasma samples. I Pharm Sci 68:1052-1054. Smith SE, Rawlins MD (1974) Prediction of drug oxidation rates in man: Lack of correlation with serum gamma-glutamyl transpeptidase and urinary excretion of o-glucaric acid and 6 B-hydroxycortisol. fur / C/in Pharmaco/7:71-75. Sotaniemi EA (7973) Effects of drug pretreatment on antipyrine levels in blood and tissues: An example of multiple drug interactions. Pharmacology 10:306-316. Statland BE, Astrup P, Black CH, Oxholm E (1973) Plasma antipyrine half-life and hepatic microsomal antipyrine hydroxylase activity in rabbits. Pharmacology 10:329-337. Stevenson IH (1977) Factors influencing antipyrine elimination. Br 1 C/in Pharmacol4:261-265. Van Boxtel CJ, Wilson JT, Lindgren S, Sjoqvist F (1976) Comparison of the half-life of antipyrine in plasma, whole blood and saliva of man. Eur j C/in Pharmacol9:327-332. Van Peer A, Belpaire F, Bogaert M (1978) Pharmacokinetics of drugs in rabbits with experimental acute renal failure. Pharmacology 17:307-324. Vesell ES, Lee C], Passananti GT, Shively CA (1973) Relationship between plasma antipyrine halflives and hepatic microsomal drug metabolism in dogs. Pharmacology 10:317-328. Vesell ES, Passananti GT (1973) Inhibition of drug metabolism in man. Drug ~etab D;spos;t;on 1:402-410. Vestal RE, Norris AH, Tobin JD, Cohen BH, Shock NW, Andres R (1975) Antipyrine metabolism in man: Influence of age, alcohol, caffeine, and smoking. C/in Pharmacof Ther 18:425-432. Welch, RM, De Angelis RL, Wingfield M, Farmer TW (1975) Elimination of antipyrine from saliva as a measure of metabolism in man. C/in Pharmacol Ther 18:249-258. Yoshimura H, Shimeno H, Tsukamoto H (1968) A new metabolite of antipyrine. Biochem Pharmacof 17:1511-1516.
D.M.
CHAMBERS AND G.C.
The application of antipyrine is described
JEFFERSON
of a gas-liquid in plasma
chromatographic
for the estimation
and is compared
(GLC) method
of antipyrine
with previous
to measurements
half-life
GLC methods.
(T,,J
in the rabbit
It has been established
that the estimation of antipyrine can be achieved with acceptable precision and that rabbits can act as their own controls in the study of factors that might alter T112. lproniazid phosphate, 50 mg/kg IP, was shown to produce a mean increase of 170% in Tli2. Key Words:
Antipyrine;
Biotransformation;
Iproniazid;
Rabbit
INTRODUCTION Antipyrine half-life (T,,J estimations have been widely used to assess the effect of factors that influence hepatic oxidative drug-metabolizing enzymes. These factors include drugs that cause inhibition or induction of these enzymes and a number of diseases (Stevenson, 1977). Whilst T,,2 has been measured extensively in man, animal studies have also made an important contribution to this field. Measurements of T,,2 have been undertaken in a variety of animal species including mouse (Quinn et al., 1958; Chambers and Jefferson, 1977), rat (Quinn et al., 1958; Sotaniemi, 1973; Aarbakke, 19781, dog (Vesell et al., 1973; Kampffmeyer, 19741, rhesus monkey (Branch et al., 19741, baboon (Down, 1976), horse (Powis and Snow, 19781, and even the quokka, a marsupial (McManus and Ilett, 1979). In general the antipyrine is estimated from blood samples although Welch et al. (1975) carried out estimations using rat saliva. For the smaller species, mouse and rat, T,,* estimates often rely on data from pooled blood samples whereas the larger species, by sequential blood sampling, allow a T 112 estimate from each animal. However, T,,2 has been estimated from individual rats, Bakke et al. (1974). It is surprising that there have been few measurements of T7,2 in vivo in the rabbit (Quinn et al., 1958; Statland et al., 1973; Van Peer et al., 1978; McManus and Ilett, 1979) as sequential blood sampling is eminently suitable in this species and because each animal might be able to act as its own control in studies of factors likely to change T,,*. Statland et al. (1973) and Van Peer et al. (1978) used rabbits as their own From the Pharmacology Section, Department of Pharmacy, Heriot-Watt University, 79 Grassmarket, Edinburgh, Scotland. Address reprint requests to D.M. Chambers, Pharmacology Section, Department of Pharmacy, Heriot-Watt University, 79 Grassmarket, Edinburgh EHI 2HJ, Scotland. Received March 1980; revised and accepted May 1980. 59 Journalof
Pharmacological
0 1981 Elsevier
North
Methods
Holland,
5, 59-66 f1981)
Inc., 52 Vanderbilt
Avenue, New York, NY 10017
OlMJ-5402/81/0059008/$02.50
60
D.M. Chambers and G.C. Jefferson controls method.
although their data gave little information about the precision of the Furthermore, their results did not show any significant change in T,/2 after
exposure before
of the animals
to certain
metabolic
studies,
monstrable
further
lengthening
was required.
Information
Therefore,
Most
workers
on the precision
of T 1,2 in the presence
It is known
that in the rabbit
antipyrine is almost exclusively zymes prior to elimination. Brodie
pretreatments.
the use of TT,* in the rabbit can be recommended
have estimated
antipyrine
et al. (1949) or the gas-liquid
et al. (1973) or Lindgren
chromatographic
tographic
(Shargel
and
of an established
and the de-
enzyme
et al., 1968),
by oxidative
Occasionally
inhibitor
as in man,
drug-metabolizing
by the spectrophotometric
chromatographic
et al. (1974).
thin-layer
that
of the method
(Yoshimura
metabolized
we considered
for drug interaction
method
(GLC) methods
radiometric
(Bakke
enof
of Prescott et al., 1974),
(Welch et al., 1975) and high-pressure liquid chromaet al., 1979) methods have been used. The present work uses a
GLC method based on that previously reported (Chambers and Jefferson, 1977) and which in turn was modified from that of Prescott et al. (1973). By contrast all of the previous metric
antipyrine method
T,,* determinations
except McManus
in the rabbit
and llett
have used the spectrophoto-
(1979) who used the radiometric
method.
METHODS Male New Zealand on commercial
white
rabbits
rabbit pellets
weighing
(McGregor)
rabbits were started at 09 hours 00 minutes at 21 *
between
3 and 4 kg were maintained
and water ad libitum.
All experiments
with the ambient temperature
on
maintained
1°C.
Antipyrine
T,,, Estimations in the Rabbit
In each rabbit antipyrine Tqr2 was estimated interval of between 21 and 28 days. Antipyrine, jected into a marginal taken for analysis
ear vein followed
from
the marginal
on two occasions separated by an 50 mgikg in sterile water, was in-
by 1000 IU of heparin.
ear vein contralateral
40, 60, 80, 100, 120, and 140 minutes.
Blood
loss
Blood samples
to the injected
were
ear at 20,
can cause a reduction
in drug
metabolism (Cumming et al., 1971) and in the present work, the total loss was 5 ml/ kg, a volume thought unlikely to have caused a reduction in antipyrine metabolism. A subsequent
study
on metabolic
inhibition
was performed in which three rabbits (control) followed T ,,2 estimation
received pretreatment with saline prior to the first by iproniazid phosphate (50 mg/kg) pretreatment All pretreatments
were given intraperitoneally
Analysis of Antipyrine To 1 ml samples (Oxford taining
Dispensor)
to the second prior
estimation.
to antipyrine.
in Plasma
of plasma
(Oxford
and 1 ml chloroform
12.5 kg phenacetin
screw-capped
prior
one hour
(Oxford
glass centrifuge
Sampler)
were added 0.2 ml of 5M NaOH
(McFarlan
Pipettor)
Smith,
as an internal
tubes with Teflon
liners
anesthetic reference
grade) constandard
in
(Sovirel).
These tubes were placed horizontally in a metabolic shaker and oscillated wise for 15 minutes at a rate of 65 per minute. The tubes were centrifuged
lengthat 3000
Antipyrine Half-life in the Rabbit g for 10 minutes. pipette
The
into individual
organic
phase was removed
tapered glass centrifuge
ness by placing the tubes
from
tubes,
each tube with
a Pasteur
and then evaporated
in a water bath at 90°C for 10 minutes.
The
to dry-
residues
were
redissolved in 20 PI of chloroform using a vortex mixer and kept on ice. Three j-4 aliquots of this solution were injected onto a 2 m x 1.75 mm o.d. glass column packed with 100-120
mesh Gas Chrom
20M. The temperature
of the column
and flame ionization
detector
rate of 90 ml/minute.
Antipyrine
antipyrine:
phenacetin
with
We
established
0.15-7.75)
that a plot
by the equation error
concentrations
gas was nitrogen
were calculated using integral
to corresponding
of peak integral concentration
y = 0.0857x-0.0265 of the regression
ratios
(2-90
ratios from
ratios
standards
pre-
of 30 pg/ml.
antipyrine:phenacetin
j.r.g/ml) was a straight
with an error coefficient,
port
used at a flow
to rabbit plasma to give a concentration
against antipyrine
standard
was 250°C. The carrier reference
pared by adding antipyrine
Q coated with 0.5% SE 30 and 0.5% carbowax was 220°C and that of both the injection
(range
line described
(S&I/~%) of 0.27 where
Sb is the
b, of the line.
Materials For injection, (Roche)
in sterile
antipyrine
(BDH
saline
(Polyfusor,
Chemicals) Boots)
in sterile
water, or iproniazid
were prepared
immediately
phosphate before
use.
RESULTS Estimations of Antipyrine Antipyrine regression
T,,? in Rabbits on Two Occasions
Tli2 was determined
for each rabbit from the slope of the least squares
line fitted to the plot of log plasma antipyrine
concentration
against time.
I 20
40
60
Time
80
100
I20
140
(min)
FIGURE 1. Antipyrine half-life in rabbit 2 (Table 3) after pretreatment with saline (0) (control) or iproniazid phosphate (O), 50 mg/kg IP. Pretreatments given one hour before antipyrine, 50 mg/kg IV. Interval between half-life determinations was 21 days.
61
62
D.M. Chambers and C.C. Jefferson TABLE 1
Antipyrine
Pharmacokinetics
in the Rabbit APPARENT
HALF-LIFE
(T,,,)
(min)
Mean SE Statistical difference between estimates (Pairedt test)
OF
(v,)
PLASMA
CLEARANCE
(PC)
(ml.min-'.kg ')
(Ikg~')
ESTIMATE? RAEEIT
VOLUME
DISTRIBUTION
ESTIMATESa
ESTIMATES”
1st
2nd
1st
2nd
1st
2nd
49.8 48.7 42.5 46.1 79.0 88.3 97.4 53.9
45.0 52.4 52.3 49.5 83.5 91.0 82.0 72.9
0.821 0.739 1.113 0.699 0.821 0.774 0.799 0.669
0.833 0.819 0.866 0.890 0.762 0.892 0.909 0.846
11.43 10.53 18.15 10.52 7.20 6.07 5.68 8.60
12.83 10.83 11.47 12.45 6.32 6.79 7.68 8.05
63.2 7.6
66.1 6.4
0.804 0.048
0.852 0.017
9.77 1.42
9.55 0.93
>0.05
>0.05
10.05
a Intervalbetween 1st and 2nd estimateswas 21-28 days.
Table 1 shows the estimates of T,,* on two occasions in each of eight rabbits. corresponding apparent volumes of distribution (V,) and plasma clearances are also given. PC was calculated (Riggs, 1963) from the equation: pC = Dose antipyrine
(mg/kg)
Co
The (PC)
.-0.693 TII2
where Dose antipyrine
=
VD
CO
and Co is the theoretical plasma antipyrine concentration at zero time. There was no significant change in T,,*, V,, or PC in the eight rabbits between estimations (p > 0.05 by paired t-test). Analysis of variance on T7,* values (Table 2) showed a significant inter-animal variation (p < 0.001).
TABLE 2 Table 1
Analysis of Variance of Antipyrine
DEGREES SOURCE
OF VARIATION
Between rabbits Within rabbits Total
SUM
OF SQUARES
5214.97 385.01 5599.97
f = 15.48 (d.f.= 7,8);p < 0.001.
FREEDOM
7 8 15
r,,, Results from
OF MEAN
SQUARE
745.00 48.13
Antipyrine Half-life in the Rabbit TABLE 3
Effect of lproniazid
Phosphate 50 mg/kg IP on Antipyrine Pharmacokinetics APPARENT
HALF-LIFE
(T,,z)
(min)
___~ 1 2 3
OF
(vo)
PLASMA
CLEARANCE
(ml.min-
(Ikg-‘1 ESTIMATE?
ESTIMATESd RABBIT
VOLUME
I~ISTRIBUTION
(PC)
‘.kg- ‘1
ESTIMATES=
1st ____.~ 100.9 81.2 81.6
2nd
1st
2nd
1st
2nd
199.4 143.3 168.2
0.678 0.732 0.662
0.636 0.697 0.432
4.72 6.25 5.62
2.21 3.37 1.78
87.9 6.5
170.3 16.2
0.691 0.021
0.588 0.080
5.53 0.44
2.45 0.47
Mean SE Statistical differences between estimates (Paired t test)
>0.05
CO.05
CO.05
a Interval between 1st and 2nd estimates was 21-28 days.
Effects of lproniazid
on Antipyrine
Pharmacokinetics
The effect of iproniazid phosphate 50 mg/kg IP in three rabbits is shown in Table 3. There was a significant lengthening of T,,2 and a corresponding reduction in PC. Figure 1 shows the change in T1,2 after iproniazid in one of the rabbits. The percentage increase of T1,2 in the three rabbits after iproniazid ranged from t-76.6% to +106.1% (mean +93.4%). The error of each regression line from which T,,2 was calculated was determined. The mean error (S&b%) ? SE from all the estimates shown in Tables 1 and 3 was 6.3 + 0.58% (n = 22). DISCUSSION GLC methods are widely used for the analysis of antipyrine in body fluids and, compared with the spectrophotometric method of Brodie et al. (1949), they have the advantage that the presence of metabolites does not contribute to antipyrine estimations (Vesell and Passananti, 1973). The experimental conditions under which the GLC methods are used have varied widely. In our work the estimation of antipyrine concentrations from integral ratios antipyrine:phenacetin represents a departure from the use of peak height ratios by previous workers (Prescott et al., 1973; Huffman et al., 1974; Chambers and Jefferson, 1977). The relationship between our peak integral ratios and antipyrine concentration is described by a straight line with an error of 0.27%. Details of the precision of such standard curves has not been given in previous work using peak heights. The method we adopted for shaking during the antipyrine extraction avoids the problem of emulsion formation at the water:chloroform interface that was reported by Prescott et al. (1973) and that we had previously seen (Chambers and Jefferson, 1977). This had caused occasional difficulties in analysis. Huffman et al. (1974)
63
64
D.M. Chambers and C.C. Jefferson claimed from
that erratic
a double
single
results
were obtained
extraction
extraction
with
here only
if emulsions
chloroform
were formed.
(Chambers
caused a 3% reduction
The
and Jefferson,
in extraction
change
1977) to the
efficiency
with
no
loss of precision. From 22 antipyrine TIr2 estimates a mean extraction efficiency of 88 t 1.6% SE was achieved. The extraction efficiencies were calculated from a comparison
of the peaks obtained from extracted antipyrine
plasma) with
peaks obtained
phenacetin preferable
in chloroform. antipyrine
(1973).
latter
The
The
recovery
solution
procedure
solution
a known
measure
of antipyrine
extracted fails
to measure
deviation
from
kg with
an error
allowing
with the 5 kg/ml a sensitivity viation
of 9.3%
detection
of Prescott
to 0.5 kg/ml
of the Lindgren
antipyrine
the
using
or within
loss of antipyrine
from
work
of this
assess
of the species
the method.
differences
known
IP. This
compares work
the first
treatment
well with
(Table
I),
with
the values
values
in seven
no evidence
imme-
was no measurable of antipyrine
has not yielded
and
sufficient the rabbit
of certain compounds given antipyrine
of 63.2 and 66.1 minutes
that show
et al., 1976)
et al. (1958) included rabbits
de-
Subsequent
for antipyrine
amounts
by others,
Quinn
kg/ml.
There
that
concentration
(Van Boxtel
analysed
in the metabolism
a mean T,,2 of 63 minutes
present
method
at -20°C.
rabbit plasma containing
compared
as the standard
in the antipyrine
were either
days after storage
stored at -20°C over a period of 82 days. Work on T,,* in the rabbit previously reported to fully
of 0.5 kg/ml
in the range 10.1-15.1
a modification
fourteen
concentration
from the rabbit suggest
especially
method was as high as 10.1%
produced even larger errors. Plasma samples in the present
in a study
by either
et al. (1974) was of increased
concentrations
is not required,
pg/ml and only fell to 3.9%
determined
retained
to
et al.
4.4%, at the 5 kg/ml concenof 4.4% for the concentration
et al. (1973). Our analyses
group
data on which
plasma given relative
over the antipyrine
of Lindgren
of antipyrine
range 0.5-5.0
diately
was
at 5 kg/ml.
work
by this
and
extraction
as used by Prescott
antipyrine
for our method
claim made for the CLC method
sensitivity,
of antipyrine
of antipyrine
or by the plasma.
mean standard
The
(30 t_r.g/mlin rabbit
solution
simultaneously
range S-60 kg/ml was 3.4% with the greatest error, tration. Prescott et al. (1973) reported a mean error range S-50
standards
unextracted
It was felt that this
to the percentage
an aqueous aqueous
from
and
50 mg/kg
determined
in the
for enzyme-induction
after
antipyrine.
Repeated measurements of antipyrine TI,2 were made by Statland et al. (1973) in each of six rabbits after antipyrine doses of 50 mg/kg IM on days 1, 8, and 14. The results show little change in the mean TIlz values on the three occasions, being 60, 65.4 and 65.4 minutes respectively. If the percentage changes in TT12from first to second and first to third determinations are calculated for each rabbit from these results, it is found that they range from -23% to +57% (mean +15.0%) and from -25%
to +76%
(mean
+18.7%),
respectively.
In the present
work
the correspond-
ing changes from Table 1 range from - 15.8 to + 35.1% (mean + 7.1%). was given by Statland of the precision of individual T,,* determinations Furthermore
their
regression
lines
of log antipyrine
concentrations
No estimate et al. (1973). on time were
Antipyrine
Half-Life in the Rabbit
determined subjectively. It was noted that they only took blood samples at 80, 160, and 240 minutes after antipyrine injections compared with the seven samples taken over 140 minutes in experiments reported here. Van Peer et al. (1978) showed a change in mean T,,2 in five rabbits given saline from 73.8 to 72 minutes over a period of five days. These T1,2values again compare well with those quoted above. As no individual T,,2 values were given by Van Peer et al. (1978) and as there is no information on the errors of their TI12regression lines it is again difficult to gain information on the precision of the method. Our mean error of 6.3% for the regression lines of log antipyrine concentration on time suggests that our analytical performance is sound (Robinson, 1971) and compares well with a value of 7.9% reported from work in man (Vestal et al., 1975). The present T,,2 results indicate that intra-animal variation is less than inter-animal variation (Table 2), a comparable finding in respect of antipyrine T,,2 in man has been reported by Lindgren et al. (1974). As well as antipyrine Tl12 values, we have calculated the corresponding PC values in the rabbit. Smith and Rawlins (1974) discussed the use of PC measurements as an alternative to T,,2 for estimating drug metabolism. They claim that this use of T ,,2 was valid only if it could be assumed that the V, was constant in different individuals. We have, however, used each rabbit as its own control and shown that V, in a given rabbit had not altered between T,,, estimations (Table 1). Furthermore, a correlation was established in the rabbit between plasma antipyrine T 1,2 and liver microsomal antipyrine hydroxylase activity by Statland et al. (19731, who suggested that T ,,* measurements serve as a reliable index of antipyrine hepatic metabolism rather than its disposition or renal excretion. These findings, together with evidence of a lack of enzyme induction in the present work, indicate that with a suitable interval between determinations rabbits can act as their own controls in the investigation of potential factors causing variation in antipyrine Tl,*. Potential factors affecting T,,, investigated in the rabbit included carbon monoxide (Statland et al., 1973) and chemically-induced renal failure (Van Peer et al., 1978), both of which were without effect on T,,2, but there have been no reported investigations in the presence of drugs. We have shown in the rabbit that iproniazid, a compound that is known to inhibit hepatic mixed function drug-metabolizing enzymes (Laroche and Brodie, 1960), produced a marked lengthening of T,,* with a corresponding reduction in its PC and our results suggest that the use of antipyrine Tl,2 determinations in the rabbit may be of value in studies on oxidative drug metabolism. We gratefully acknowledge
the gift of iproniazid phosphate from Roche.
REFERENCES Aarbakke J (1978) Disposition and oxidative metabolism of antipyrine in the rat. Acta Pharmacol et Toxic0143
Bakke, OM,
:64-68. Bending
M, Aarbakke
J, Davies DS
(1974) ‘%Antipyrine as a model compound in the study of drug oxidation and enzyme induction in individual surviving rats. Acta Pharmacol et Toxicol35:91-97.
65
66
D.M. Chambers and G.C. Jefferson Branch RA, Shand DG, Wilkinson GR, Nies AS (1974) Increased clearance of antipyrine and dpropranoloi after phenobarbital treatment in the monkey. 1. Clin fnvest 53:1lOl-1107. Brodie BB, Axelrod I, Soberman R, Levy BB (1949) The estimation of antipyrine in biological materials. /. Bio/ Chem 179:25-29. Chambers DM, Jefferson GC (1977) Some observations on the mechanism of benzodiazepine-barbiturate interactions in the mouse. f?r j Pba~maco/ 60:393-399. Gumming, JF, McClung HW, Mannering G] (1971) The effect of hemorrhage on the rate of hexobarbital biotransformation in the dog. / Pharmacol Exp Ther 178:595-601. Down WH (1976) Species differences in drug metabolism; in vivo parameters of hepatic drugmetabolising enzyme activity in the baboon. In Microsomes and Drug Oxidation. Ed., V Ullrich. 0xford:Pergamon Press, pp. 516-519. Huffman DH, Shoeman DW, Azarnoff DL (1974) Correlation of the plasma elimination of antipyrine and the appearance of 4-hydroxyantipyrine in the urine of man. Biochem Pharmaco~ 23:197-201. Kampffmeyer HG (1974) Metabolic rate of phenacetin and of paracetamol in dogs before and after treatment with phenobarbital or SKF 525 A. Biochem Pharmacol23: 713-724. Laroche M, Brodie BB (2960) Lack of relationship between inhibition of monoamine oxidase and potentiation of hexobarbital hypnosis. f Pharmacol Exp Ther 130:134-137. Lindgren S, Collste P, Norlander B, Sjoqvist F (1974) Gas chromatographic assessment of the reproducibility of phenazone plasma half-life in young health volunteers. Eur/ C/in Pharmaco/7:381-385. McManus ME, llett KF (1979) Comparison of rate of hepatic metabolism in vitro and half-life for antipyrine in vivo in three species. Xenobiotica 9:107-118. Powis G, Snow DH (1978) The effects of exercise and adrenaline infusion upon the blood levels of propranolol and antipyrine in the horse. / Pbarmaco/ Exp 7’her 205:725-731, Prescott LF, Adjepon-Yamoah KK, Roberts E (1973) Rapid gas-liquid chromatographic estimation of antipyrine in plasma. / Pharm Pharmacol 25:205-207. Quinn GP, Axelrod J, Brodie BB (1958) Species, strain and sex differences in metabolism of hex-
obarbitone, amidopyrine, antipyrine and aniline. Biochem Pharmacol1:152-159. Riggs DS (1963) 7’he ~afhema~ica/ Approach to fhys;o/ogicai Problems. Baltimore: Williams and Wilkins, p. 127. Robinson R (1971) Clinical Chemistry mation. London: Griffin, pp. 1-18.
and Auto-
Shargel L, Cheung W, Yu ABC (1979) High-pressure liquid chromatographic analysis of antipyrine in small plasma samples. I Pharm Sci 68:1052-1054. Smith SE, Rawlins MD (1974) Prediction of drug oxidation rates in man: Lack of correlation with serum gamma-glutamyl transpeptidase and urinary excretion of o-glucaric acid and 6 B-hydroxycortisol. fur / C/in Pharmaco/7:71-75. Sotaniemi EA (7973) Effects of drug pretreatment on antipyrine levels in blood and tissues: An example of multiple drug interactions. Pharmacology 10:306-316. Statland BE, Astrup P, Black CH, Oxholm E (1973) Plasma antipyrine half-life and hepatic microsomal antipyrine hydroxylase activity in rabbits. Pharmacology 10:329-337. Stevenson IH (1977) Factors influencing antipyrine elimination. Br 1 C/in Pharmacol4:261-265. Van Boxtel CJ, Wilson JT, Lindgren S, Sjoqvist F (1976) Comparison of the half-life of antipyrine in plasma, whole blood and saliva of man. Eur j C/in Pharmacol9:327-332. Van Peer A, Belpaire F, Bogaert M (1978) Pharmacokinetics of drugs in rabbits with experimental acute renal failure. Pharmacology 17:307-324. Vesell ES, Lee C], Passananti GT, Shively CA (1973) Relationship between plasma antipyrine halflives and hepatic microsomal drug metabolism in dogs. Pharmacology 10:317-328. Vesell ES, Passananti GT (1973) Inhibition of drug metabolism in man. Drug ~etab D;spos;t;on 1:402-410. Vestal RE, Norris AH, Tobin JD, Cohen BH, Shock NW, Andres R (1975) Antipyrine metabolism in man: Influence of age, alcohol, caffeine, and smoking. C/in Pharmacof Ther 18:425-432. Welch, RM, De Angelis RL, Wingfield M, Farmer TW (1975) Elimination of antipyrine from saliva as a measure of metabolism in man. C/in Pharmacol Ther 18:249-258. Yoshimura H, Shimeno H, Tsukamoto H (1968) A new metabolite of antipyrine. Biochem Pharmacof 17:1511-1516.