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From Lehrstuhl für Pharmakologie und Rechenzentrum der U n i v e r s i t ä t Regensburg and Urologische K l i n i k ,
Elisabeth-Krankenhaus, Straubing (Federal Republic of Germany)
High-performance Liquid Chromatography Analysis of Mezlocillin,
Piperacillin, their Degradation Products, and of Ioxitalamic Acid
in Plasma and Urine of Healthy Volunteers
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By F. Rees , K . G . Naber , B. Bartoschik-Wich , P. Stockmann , G . P. M e y e r , and H . Grobecker
Summary: In plasma and urine of 10 healthy volunteers
after intravenous administration
of 4 g mezlocillin and piperacillin,
respectively, the parent compounds as well as
degradation products were assayed by high-performance
li-
1
quid chromatography.
Ioxitalamic acid, a renal contrast medium, was administered simultaneously, in order to measure
the glomerular filtration rate, and to control the collection of
24-h urine. As metabolite of mezlocillin the
corresponding
penicilloic acid only was found, whereas in the case of piperacillin a further degradation product was observed. Half of
the doses given was recovered in the urine as unchanged
drugs, and in addition 5-10% as metabolites. No differences
were found in the pharmacokinetic
behaviour of both antibiotics.
Zusammenfassung:
Hochdruckflüssigkeitschromatographische Bestimmung von Mezlocillin, Piperacillin, ihrer Abbauprodukte und von loxitalaminsäure
im Plasma und Urin gesunder Probanden
Nach intravenöser Applikation von je 4 g Mezlocillin bzw.
Piperacillin
wurden mit Hilfe der
Hochdruckflüssigkeits-
Key words: Acylureidopenicillins • Diagnostics • Ioxitalamic
pharmacokinetics • Piperacillin, clinical
pharmacokinetics
acid, clinical pharmacokinetics
1. Introduction
M e z l o c i l l i n (6-[D-2-(2-oxo-3-mesylimidazolidine-1 -carboxamido)-2-phenylacetamido]penicillanic acid) and piperacill i n (6-[D(-)-a-(4-ethyl-2,3-dioxo-l-piperazinyl-carbonyl-amino)-2-phenylacetamido] penicillanic acid) are acylureido
penicillins with broad antibacterial activity and with only
slight differences between their structures (Scheme 1). Penicillins are in general partially metabolized, m a i n l y to
inactive penicilloic acids [1]. T h i s also was shown for mezloc i l l i n , but in vitro degradation o f mezlocillin in plasma
specimens may have influenced some results, leading to false
high concentrations o f mezlocillin penicilloic acids [3]. So
far, no metabolites o f piperacillin have been detected i n
plasma, though some inactive degradation products have
been observed in urine and bile [2].
R-CH-C0-NHv*^S
i
Ph
chromatograpihie
die Muttersubstanzen
sowie
Abbauprodukte dieser Ureidopenicilline
im Plasma und Urin von 10
freiwilligen Prcobanden bestimmt. Um die Nierenfunktion
zu
überprüfen unid die vollständige
Urinsammlung
über 24 h zu
kontrollieren, (erhielten die Versuchspersonen simultan
loxitalaminsäure,
ein Kontrastmittel,
infundiert.
Als einziger
Metabolit
vom Mezlocillin
wurden geringe Mengen der
entsprechendem Penicilloinsäure
gefunden. Im Falle von Piperacillin
wuirde ein weiteres Abbauprodukt
beobachtet.
Etwa die Hälfte der gegebenen Antibiotika-Dosen
wurde unverändert im Urin wiedergefunden, zusätzlich
noch 5-10%
in Form von Abbauprodukten.
Bei den
pharmakokinetischen Parametern
beider Penicilline
wurden keine signifikanten Untersschiede festgestellt.
2.2. Administration of antibiotics
After giving infformed written consent the subjects received 4 g o f
mezlocillin and after 6 weeks 4 g o f piperacillin. Concurrently, the
subjects receivecd 5 m l Telebrix 300®, equivalent to 2.4 g ioxitalamic
acid, as an inteirnal standard for comparison o f the two antibiotics.
The injection o f this contrast m e d i u m allows to measure the renal
function as glormerular filtration rate, and to control the collection o f
the 24-h urine. T h e antibiotics and ioxitalamic acid were dissolved
together i n 50 m l water for injections a n d infused intravenously
within 30 m i n ait a constant rate.
R-CH-CO-NH
.
R-CH-C0-NH
S
-CO
OH
C00H
clinical
and 188 c m (miedian 174 cm). T h e health o f the volunteers was
established fromi their medical history, physical examination and laboratory screeniing (differential blood count, platelet count, serum
creatinine, S G O ) T , S G P T , G G T and urine analysis). N o subject was
hypersensitive tco penicillins o r contrast media. Pregnancy was excluded by the retgular use o f contraceptives.
• n2 L
rs
• Mezlocillin,
:
X
Ph
C00H
C:OOH
0
R =
R
= • -N
N- Et
1
: piperacillin
+ H
H 29 0
0
Ho-U
> -HN
\
mez.
penicilloic
acid
pip.
penicilloic
acid
mez
penilloic
acid
o
(
/
N- Et-
6 - ( D - ( - } - * - { 3 - [ 2 - ( N-efhy I -N-oxalamino ) ethyl ] ureido} - «t - phenylaceramido ]
penicilloic
acid
Scheme 1: C h e m i c a l structures o f m e z l o c i l l i n , p i p e r a c i l l i n , and degradation p r o d u c t s .
The a i m of the present study was to establish the pharmacokinetics of mezlocillin and piperacillin after intravenous i n fusion o f 4 g over 30 m i n , and to quantify their probable
main metabolites, the corresponding penicilloic acids. F o r
analysis a liquid chromatographic method was chosen i n stead of bioassay, because these ring-open penicillin derivatives are not antibacterially active. Care was taken with the
sampling and storage of plasma samples because o f in vitro
instability of mezlocillin and other penicillins i n body fluids
[3].
2. Material and methods
2.3. Blood and murine collections for assay
Venous blood ssamples (6 to 8 ml) were withdrawn into heparinized
syringes from a. contralateral arm vein through an indwelling needle
before and at s p e c i f i e d intervals after infusion. These samples were
taken at 15 m i m after start of the infusion, at the end of infusion and
at 10, 20, 30, 4:5, 60, 9 0 m i n , 2, 3, 4, 6 and 8 hours thereafter. B l o o d
samples were ccentrifuged at 4 °C within 30 m i n and the plasma was
stored at - 7 0 ° C until analysis. U r i n e samples were collected before
the start o f the experiments and during the periods 0 to 4.5, 4.5 to
8.5, 8.5 to 12.55, a n d 12.5 to 24.5 h after the start o f infusion. T h e
p H (ranging f r o m 5.0 to 6.5) and volumes of all urine samples were
measured, and the specimens were stored at - 7 0 °C. T h e excretion
of creatinine i m the fractionated urine collections over 24 h was assayed by the auitoanalyser modification o f the method o f JafTe (4).
2.1. Subjects
2.4. Chromatographic assay
The study included 10 healthy subjects (5 male, 5 female, aged between 22 and 34 years, median 29 years). T h e i r weights ranged between 50 and 86 kg (median 66 kg), and their heights between 154
2.4.1. Reagents* and chemicals
M e z l o c i l l i n witth the respective penicilloic acid and penilloic acid,
piperacillin wiith its penicilloic acid and ioxitalamic acid (5-acet-
a m i d o - N - ( 2 - h y d r o x y - e t h y l ) - 2 , 4 , 6 - t r i i o d o i s o p h t a l a m i c acid) were
supplied by the respejctive manufacturers. A s diagrnostic agent rrueglumine ioxitalamate ( T e l e b r i x 3O0®; manufacturer: Byk G u l d e n ,
Konstanz, F R Germainy)) was used.. Acetonitrile ( H P L C grade S) was
purchased from Zinsser, F r a n k f u r t / M a i n ( F R G e r m a n y ) , tetrabutyla m m o n i u m hydrogensuLfate from F l u k a , N e u U l m ( F R G e r m a n y ) .
A l l other chemicals (;anaJytical grade) were o b t a i n e d from E . M e r c k ,
Darmstadt ( F R G e r m a n y ) . W a t e r was purified w i t h a M i l l i - Q water
purification system ( M i l L i p o r e , E s c h b o r n , F R Germiany).
Stock solutions o f the antibiotics, their metabolites and of i o x i t a l amic acid were prepared in waiter to yield finail concentrations
of 1 m g / m l , and stored in aliquots at - 2 0 °C. T h e stock solutions
were than diluted w i t h drug-free p l a s m a to p r o v i d e assay standards
of 50 / i g / m l for mez:locillin and p i p e r a c i l l i n and 2 0 / i g / m l for the
penicilloic acids o f b o t h p e n i c i l l i n s . F o r checking the linearity o f the
assay dilutions o f 1 0 O - 0 . 7 8 ^g/'ml for m e z l o c i l l i n amd p i p e r a c i l l i n in
plasma and 2 0 - 0 . 6 3 /ig/'ml for m e z l o c i l l i n p e n i c i l l o i c acid were prepared. Lack o f sufficient material d i d not a l l o w to prepare a standard series o f p i p e r a c i l l i n penicilBoic acid. F o r uirine samples the
standard solutions were prepared in 50 mmol/1 s o d i u m phosphate
buffer ( p H 6.5).
2.4.2. Sample treatment
Plasma samples were treated a c c o r d i n g to a publislhed procedure [5]
with minor modifications. In brief, 200 p\ plasrma were buffered
with 200 ß\ 50 mmol/1 s o d i u m phosphate ( p H 6.(3) and deproteinized with 400 ß\ acetonitrile. T h e latter was than extracted into 2 m l
dichloromethane, amd 1 0 - 2 0 iA o f the aqueous phase, containing
the penicillins and t h e i r metabolites, were injected into the Chromatograph. U r i n e was centrifuged a n d diluted tenfold with 50 m m o l / 1
sodium phosphate buffer ( p H 6.5). A l l b i o l o g i c a l samples were
stored at - 7 0 °C (up to 7 weeks) a n d thawed i n i c e d water just p r i o r
to analysis.
2.4.3. Chromatography
T h e chromatographic system consisted o f a p u m p M 6 0 0 0 A , an automatic injector W I S P 710B (fitted with a c o o l i n g kit, in order to
maintain 8 °C for the samples), a R C S S compression m o d u l e
equipped with a cartridge (100 x 5 m m I.D.) p a c k e d with N o v a p a k ®
C-18 4 - 5 ßm silica, a fixed-wavetlength detector M 441, a data m o dule M 730 and a system c o n t r o l l e r M 720 (all fromi Waters Assoc.,
Eschborn, F R G e r m a n y ) .
T h e flow rate was m a i n t a i n e d at 1.0 m l / m i n . the resulting backpressure was 6000 k P a . T h e eluent was m o n i t o r e d at 214 n m ( Z n lamp) for the determination o f m e z l o c i l l i n and at 2129 nm (Cd-lamp)
for the determination o f p i p e r a c i l l i n . F o r m e z l o c i l l i n analysis the
mobile phase was prepared by c o m b i n i n g 760 m l o f 12.5 m m o l / l sod i u m phosphate buffer ( p H 6.8), 240 m l acetoniitrile and 150 mg
tetrabutylammonium hydrogen sulfate. T h e appairent p H was adjusted to 7.3 with 10 N s o d i u m hydroxide. Part o f the m e z l o c i l l i n
samples was analysed using a H I B A R ® c o l u m n ( 1 2 5 x 4 m m I.D.)
filled with L i C h r o s p h e r ® R P - 1 8 5 ^ m silica (E. N4erck; for m o b i l e
phase composition see F i g . 1). F o r piperacillin assay 1 g tetrabutyla m m o n i u m hydrogen sulfate was used and the p H adjusted to 6.5.
For determination o f i o x i t a l a m i c acid a H I B A R c o l u m n ( 1 2 5 x 4
m m I.D.), prepacked with L i C h r o s o r b ® R P - 1 8 5 / i m silica (E.
Merck), was used for separation. T h e flow rate w a s maintained at
1.0 m i / m i n , the back-pressure was 11000 k P a . T h e mobile phase
was a mixture of 9 1 0 m l water, 90 m l acetonitrile, 600 /u\ acetic
acid, 350 m g t e t r a b u t y l a m m o n i u m hydrogen sulfiate. The p H was
adjusted to 4.8 with 10 N s o d i u m hydroxide. T h e eluent was m o n i tored at 254 n m (Hg-lamp), the retention time olf ioxitalamic acid
was about 4.5 m i n .
2.4.4. Pharmacokinetic analysis
Plasma level data were analysed b y the open, two-compartment m o del. Tne decline in drug, plasma levels in the postimfusion phase was
fitted by a computer program for each subject u s i n g an iterative relative least-squares regression analysis. A Fortran p r o g r a m was used in
the computation. T h e basic equation o f the mathematical m o d e l
was
,-ßt
1
Cj> = A • e-* + B •
where O represents the plasma concentrations att time t after the
dose. 2 and ß ( m i m ) are h y b r i d constants o f the fast and the s l o w
disposition process, respectively, and A and B (mig/1) are the zerotime intercepts of the two components o f the biex.ponential curves.
T h e calculated pharmacokinetic constants were corrected for infusion t me [6]. A n u m b e r o f parameters have been calculated, especially following: area under the plasma concentiration-time curve
(AUG).«,): apparent steady-state volume o f d i s t r i b u t i o n (V$s), total
body :learance ( C l t ) , and t e r m i n a l plasma half-life* (U/iß)p
1
l0
k
£ 0
min
8
.£'0
U min
8
E O
Fig. 1: C h r o m a t o g r a m s o f (1) an aqueous standard solution of m e z l o c i l l i n
( M E Z , 100 ng) m e z l o c i l l i n p e n i c i l l o i c acid ( M P C : 84 ng mixture o f epimers A
and B), and m e z l o c i l l i n p e n i l l o i c a c i d ( M P L ; 150 ng mixture o f epimers A
and B), (2) a plasma blank and (3) plasma o f a volunteer 4 h after intravenous
injection (3 min) o f 5 g m e z l o c i l l i n . T h e asterisk (*) marks a i unidentified
substance that appears in treated plasma o r aqueous samples, a i d disappears
w i t h i n 2 days at r o o m temperature (see chromatogram 2). Concentrations:
M E Z = 15 / / g / m l . M P C = 2.5 ^ g / m l . A U = absorption units.
Chromatographic conditions: C o l u m n : H I B A R LiChrospher RP-18 5 ^ m ;
(125 x 4 m m I.D.); mobile phase: 2,8 g s o d i u m dihydrogen phcsphate m o n o hydrate, 255 mg t e t r a b u t y l a m m o n i u m hydrogen sulfate, 750 ~nl water, 250
m l acetonitrile, apparent p H 6.2: flow rate: 1 m l / m i n ; pressure 11000 k P a .
3. Results
3.1. Chromatography
In F i g . 1 chromatograms o f a standard mixture of m e z l o c i l lin and its degradation products, mezlocillin pen c i l l o i c and
penilloic acid, and of extracted human plasma are depicted.
Each m e z l o c i l l i n metabolite elutes as two distinct peaks because of epimerization at the carbon atom in position 5 (see
Scheme 1) [7]. The earlier eluting isomers A are the m i n o r
components in freshly prepared aqueous solutions, but become dominant when standing at r o o m temperature for
many hours as it was also described for amoxycillin p e n i c i l loic acid [8]. Isocratic separation o f all compounds w i t h i n 10
minutes, as seen i n Fig. 1, was enabled by application of reversed phase ion pair chromatography. F o r L i C h r o s p h e r R P
18 silica a good compromise between separation of m e z l o c i l l i n and its degradation products from interfer.ng plasma
components, and run time was found with 255 mg/1 tetrab u t y l a m m o n i u m hydrogensulfate in the mobile phase and
p H 6.2. For N o v a p a k C-18 silica a lower content of tetrab u t y l a m m o n i u m hydrogen sulfate (150 mg/1) and a higher
p H (7.3) proved to give better resolution (see Materials and
methods for more details). A s metabolite o f mezlocillin in
plasma and urine, we found mezlocillin penicilloic acid
only. Since it has two carboxylic groups, the retention time
is more sensitive to changes in concentration of tetrabutyla m m o n i u m salt and p H than that of mezlocillin hself.
F i g . 2 shows chromatograms of a standard mixture of pipera c i l l i n and p i p e r a c i l l i n penicilloic acid, prepared by incubation o f piperacillin with ^-lactamase, and o f plasma samples
of a volunteer after injection of 4 g p i p e r a c i l l i n . Like p e n i c i l loic acid derived from m e z l o c i l l i n , piperacillin penicilloic
acid elutes as two separated epimers, and is hardly to determine in plasma. O n the other hand, two higher unidentified
peaks appear at shorter retention times.
3.2.
Evaluation of the assay
T h e chromatographic peaks were quantitated by the area
method. The areas of the two unidentified metabolites of p i -
* v
j ~ «
25*» nm
PIP
0.002
AU
PPC
A B
X
2 2 9 nm
X
AB
AB
IIUI Ii
PPC
A B
QJ «^ 0
r
CD
U min
8
1
'.E 0
4 min
8
-
0
4 min
8
:e
0
i* min
8
Fig. 2: Chromatograms o f ( 1 ) an aqueous standard s o l u t i o n of piperacillin ( P I P , 250 ng) and p i p e r a c i l l i n penicilloic acid ( P P C , ca. 200 ng m i x t u r e o f epimers A
a n d B ; the asterisk marks an i m p u r i t y , presumably a hydrolysis product o f piperacillin), (2) a plasma blank, (3) plasma o f a v o l u n t e e r 1 h and ( 4 ) 4 h after the
end o f an intravenous infusion (30 min) o f 4 g p i p e r a c i l l i n . X marks two unidentified peaks ( A and B), probably a m i x t u r e o f epimers. concentrations: (3) P I P =
47 ßg/ml
P P C = 2,3 / i g / m l , X = 9,8 # g / m l when quantified as PIP. (4) P I P = 2,6 / / g / m l ; X = 4,8 ^ g / m l . C h r o m a t o g r a p h i c c o n d i t i o n s : see M a t e r i a l and methods. A U = absorption units.
p e r a c i l l i n were s u m m a r i z e d and quantified as piperacillin.
T h e recovery from p l a s m a was 101.1 ± 1.3% for mezlocillin
(concentration 100 /ag/ml: n = 9), for mezlocillin penicilloic
acid 1 1 0 0 . 8 ± 2 . 8 % (concentration 42 / i g / m l ; n = 9), for piperacillim 104.3 ± 4 . 0 (concentration 50 ^ g / m l ; n = 15) and for
p i p e r a c i l l i n penicilloic acid 91 to 93% (concentration 20
/ig/m.l; n = 3). T h e recovery from urine was not checked and
set 100%, as the only sample treatment step was dilution of
urine with buffer.
S o m e plasma specimens o f the mezlocillin study were determ i n e d at two different days and the following reproducibility
was found: m e z l o c i l l i n 102.7 =b4.8% (concentration range
3.95 to 328 ywg/ml), m e z l o c i l l i n penicilloic acid 1 0 7 ± 2 0 %
(concentration range 0.80 to 3.6 //g/ml). T h e precision of the
p i p e r a c i l l i n assay was checked with spiked plasma. T h e results are shown i n Table 1.
3.3. Pharmacokinetics
Fig. .3 and 5 show the mean ( ± S D ) plasma concentrations
and the cumulative urinary excretion of mezlocillin and its
ring-open metabolite, F i g . 4 and 6 the respective data of p i peracillin. In addition, the plasma concentrations of ioxital a m i c acid are depicted. In both cases, the mean plasma
concentrations o f the metabolites were always by far lower
than the values o f the respective parent compounds. A p p a r T a b l e 1: Precision o f the d e t e r m i n a t i o n o f p i p e r a c i l l i n in plasma. ( P i p e r a c i l lin a d d e d : 25, 5, 0.5 / / g / m l . )
Day
1
2
3
4
5
Mean
S D (%)
P i p e r a c i l l i n (//g/ml)
25.2
26.0
25.3
26.1
26.2
5.69
5.85
5.29
5.96
5.74
0.60
0.69
0.62
0.69
0.70
25.8
0.5(1.8%)
5.71
0.25 (4.5%)
0.66
0.05 (7.0%)
ently neither mezlocillin nor piperacillin is metabolized in
vivo to a greater extent. These findings are also illustrated in
Table 2. In both studies we found nearly identical values for
ioxitalamic acid in plasma, and in the beginning also for
mezlocillin and piperacillin. W i t h the time elapsed, the
concentrations of mezlocillin remained slightly higher than
those of piperacillin.
T h e pharmacokinetic parameters of mezlocillin, piperacill i n , and o f ioxitalamic acid are summarized in Table 3. A l l
substances exhibit distribution volumes nearly 20% of body
weight which agrees good with the extracellular space. Like
iotalamic acid [9], ioxitalamic acid shows the same plasma
clearance as inulin (ca. 120 m l / m i n ) , whereas those of mezlocillin and piperacillin are about 250 m l / m i n . O n the other
hand, the half-life of ioxitalamic acid in plasma is about 2 h,
whereas the half-lives of both antibiotics are 1 h. The urinary recovery of ioxitalamic acid within 24 h was 90% in
each study so that complete 24-h urine collection can be assumed. A l s o the excretion of creatinine was normal in all
subjects. A b o u t 50% o f the antibiotic doses given were recovered in the urine as active parent compounds, and 9% as
metabolites in the case of piperacillin and 4% in the case
of mezlocillin.
4. Discussion
4.1. Chromatographic assay
Besides the traditional bioassay, mezlocillin and piperacillin
have been also determined in biological fluids by reversed
phase H P L C [10-15]. But in all these cases the parent c o m pounds only were assayed, as the ring-open metabolites are
much more polar and elute with the front. Using gradient
elution technique mezlocillin [16], azlocillin [17], apalcillin
[18], and the respective penicilloic acids could be determined simultaneously in serum and urine. Unfortunately,
all assays are considerably time consuming. In the present
assay incorporation of ion-pair chromatography enabled iso-
T a b l e 2 : M e a n ( ± S D ) concentrations o f m e z l o c i l l i n and p i p e r a c i l l i n after infusion (30 min) o f 4 g. and o f s i m u l t a n e o u s l y administered i o x i t a l a m i c acid (2.4 g)
in p l a s m a o f t e n healthy volunteers. I T X p i p = i o x i t a l a m i c acid in the piperacillin study: I T X m e z = i o x i t a l a m i c acid in the m e z l o c i l l i n study; M E Z = m e z l o c i l lin ; P I P = p i p e r a c i l l i n .
Time
-15
0
10
20
30
45
60
90
2
3
4
6
8
1
min '
min »
min
min
min
min
min
min
h
h
h
h
h
2
ITXpip
(/ig/ml)
ITXmez
Gwg/ml)
141 ± 2 7
194 ± 5 1
142 ± 1 8
126 ± 1 6
110 ± 1 5
94.9 ± 15.9
79.5 rt 11.5
6 2 . 5 ± 9.9
49.3 ± 9.9
3 2 . 0 ± 8.2
22.1 ± 6.0
1 0 . 4 ± 3.0
5.1 ± 2.3
134 ± 4 0
187 ± 4 5
144 ± 3 5
121 ± 2 7
108 ± 2 4
91.3±17.9
7 8 . 5 ± 16.0
6I.0±12.2
4 9 . 2 ± 10.8
3 2 . 0 ± 7.5
21.1 ± 6.9
9 . 6 ± 3.9
5 . 0 ± 2.4
ITXmez
95.0
96.4
101.4
104.3
98.2
96.2
98.7
97.6
99.8
100.0
95.5
92.3
98.0
Mean ± S D
1
'
2 )
MEZ
MEZ
(jug/ml)
PIP
(A*g/ml)
200 ± 58
271
±61
198 ± 4 6
158 ± 3 9
129 ± 3 4
97.7±25.8
73.0±21.8
45.1 ± 1 6 . 5
29.5±13.4
1 2 . 5 ± 4.7
6.0 ± 2.4
1 . 6 ± 0.8
0.6 ± 0.4
198 ± 4 2
272 ± 7 5
187 ± 3 3
148 ± 2 6
118 ± 2 6
87.7±22.5
65.2±14.5
39.6±11.2
24.1 ± 8.4
I 1 . 5 ± 5.8
5 . 5 ± 3.1
1 . 5 ± 0.6
0 . 6 ± 0.2
ITXpip
100 (%)
PIP
110.0
99.6
105.9
106.8
109.3
111.4
112.0
113.9
122.4
108.7
109.1
106.7
100.0
108.2±6.2
98.0±3.1
15 m i n after the start of infusion (the antibiotics were infused at a constant rate over 30 min).
A t the end of infusion.
Table 3 : C o m p a r a t i v e p h a r m a c o k i n e t i c parameters (mean ± SD) o f m e z l o c i l l i n (4 g), p i p e r a c i l l i n (4 g), a n d simultaneously administered i o x i t a l a m i c acid
(2.4 g) given as short intravenous infusion (30 m i n ) to 10 healthy volunteers. A b b r e v i a t i o n s : I T X m e z = i o x i t a l a m i c acid i n the m e z l o c i l l i n test; I T X p i p = ioxitalamic acid in the piperacillin test; M E Z = m e z l o c i l l i n ; P I P = p i p e r a c i l l i n : Vss = steady-state v o l u m e o f d i s t r i b u t i o n : A U C = area under the plasma concentration-time curve; C l t = total body clearance: iß - terminal plasma half-life; Vss% b.w. = Vss in percentage o f body weight; Clt 70 kg = Clt n o r m a l i z e d to 70 kg
body weight; U o - 2 4 h = 24-h u r i n a r y recovery.
ITXmez
ITXpip
MEZ
PIP
Uo-24 h
(% of dose)
])
Vss
Vss% b.w.
(1)
(%)
AUC
(mg/1 h)
Clt
(ml/min)
C l 7 0 kg
( m l / m i n / 7 0 kg)
(min)
15.5 ± 3.2
15 + ? 6
14.3 + 3.8
14.5±2.7
23.5±3.6
23.3±4.5
21.8 + 5.1
22.2±4.7
349±71
359 ± 5 9
303 ± 7 6
282±6l
118±22
1I4±18
231 ± 5 0
246 ± 5 0
127±27
123±27
247 ± 5 2
262 ± 5 4
106 ± 1 5
107 ± 1 4
63.9±11.0
66.4±13.0
t
93.1 ± 7.1
87.2±11.4
4 9 . 6 ± 4.7 )
4 9 . 6 ± 3.8 )
') 24-h creatinine excretion: m e z l o c i l l i n study (1801 ± 5 3 0 mg (male): 1 2 1 5 ± 6 0 9 (female)). P i p e r a c i l l i n study ( 2 1 0 2 ± 130 mg (male). 1 3 0 9 ± 4 1 2 (female)).
> In a d d i t i o n : 4.1 ± 1.0% as m e z l o c i l l i n p e n i c i l l o i c acid.
» In addition: 4 . 2 ± 0 . 7 % as p i p e r a c i l l i n p e n i c i l l o i c acid, and 5 . 0 ± 1.5% as c o m p o u n d X .
2
3
2
3
cratic determination of mezlocillin, piperacillin, and their
probable main metabolites within 10 m i n at a moderate flow
rate.
F r o m the present investigation it results that m e z l o c i l l i n and
piperacillin are degraded only to a very small extent to the
respective penicilloic acids after intravenous injection (Fig.
3 and 4). Moreover, it seems doubtful whether the measured
concentrations of the penicilloic acids are caused by enzymatic metabolization, or mainly by mere chemical hydrolysis. The instability of these and other penicillins in standing
plasma specimens was several times reported, and even at
- 1 8 °C 20% degradation o f mezlocillin in plasma within 6
weeks was observed, whereby half of it was found in form of
mezlocillin penicilloic acid [3]. Therefore, the detection of
large amounts of mezlocillin penicilloic acid in any body
fluid specimen may indicate rather in vitro degradation of
the parent compound than in vivo metabolism. In such
cases, false low concentrations of mezlocillin are measured,
and determination of degradation products can prove it (cf.
[3]). In accordance with results examined by G a u and Förster [16] we did not find mezlocillin penilloic acid in plasm a and urine, and indeed, the detection o f penilloic acid in
body fluids [19] may have been an artefact of the analytical
procedure [3].
Even quantitative disappearance of piperacillin from plasma
specimens was stated when stored for several weeks at
- 2 0 °C ([10]; unfortunately, though H P L C was used, no attempt was made to look for degradation products). O n the
other hand, at - 7 0 °C both antibiotics proved to be stable [3,
10].
In our study, a l l specimens were stored at - 7 0 °C no longer
than 7 weeks so that stability of piperacillin should be guaranteed. Moreover, we found only small amounts of pipera c i l l i n penicilloic acid, the supposed m a i n degradation product i n stored plasma. But beyond that, two unidentified signals appeared in the chromatograms o f plasma and urine
(Fig. 2, chromatograms 3 and 4, peaks X - A and X - B ) .
O n following reasons we believe the signals may refer to
6- [ D - (-)-a- {3- [2- ( N - e t h y l - N - o x a l a m i n o ) ethyl] ureido}-#phenylacetamido] penicilloic acid (Scheme 1), denoted X in
Fig. 1, which is partially epimerized at the carbon atom in
position 5, and elutes therefore as two distinct peaks. First,
the peak/height ratio 229 to 254 n m (Fig. 2) o f X - A and X - B
is similar to those of piperacillin and piperacillin penicilloic
acid which fits the proposed structure. Second, with time the
relative amount of the faster eluting peak increases in
accordance with findings i n the case of the penicilloic acids
of mezlocillin and apalcillin ([18]; unfortunately, piperacill i n penicilloic acid was hardly to quantify in plasma, a l l the
more since the isomer A was often covered by an endogenous compound). T h i r d , the same splitting of the d i o x o p i perazine ring was foundi i n the case of cefoperazone [20].
T h e Baternan-function-like concentration-time course of
compound X in plasma (Fig. 4) demonstrates it to be formed
in vivo, presumably as a metabolite of piperacillin penicilloic acid. However, the identity of the compounds X - A and
X - B remains to be elucidated.
4.2. Comparative pharmacokinetics of mezlocillin and
piperacillin
T h e study was not designed in a randomised cross-over fashion but the analysis of the pharmacokinetic parameters of
ioxitalamic acid in both tests (Table 3) revealed nearly identical mean values so that similar conditions can be assumed
to exist for both antibiotics mezlocillin and piperacillin. A l l
their mean pharmacokinetic parameters differed not more
than 10% from each other which is within the analytical er-
ror, and in accordance with published data [21, 22]. W e observed also the same urinary recovery of both antibiotics,
about half of the dose given, which was to be expected for
mezlocillin only [3, 16, 21]. Tjandramaga [22] found 80% of
piperacillin in the urine when a 4 g intravenous dose was
given. T h i s is in clear contrast to our results. O n the other
hand, a range of 4 0 - 7 0 % renal excretion of piperacillin is
also reported (cf. [23, 24]). So, for piperacillin a very variable range of renal excretion is found. O u r data are at its
lower margin, but in good agreement with the renal excretion of the other acylureido penicillins, mezlocillin [3. 16,
21] and azlocillin [17], which exhibit very similar chemical
structures.
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Acknowledgements
W e wish to thank D r . A . Huber from Bayer A G . M u n i c h , for samples o f mezlocillin, and D r . M . H e l m e r k i n g from C y a n a m i d - L e e e i i e .
Wolfratshausen, for samples of piperacillin.
For the authors: Prof. Dr. med. H . Grobecker, Lehrstuhl für Pharmakologie der Universität Regensburg. U n i v e r s i t ä t s s t r a ß e 31,
D-8400 Regensburg (Federal R e p u b l i c of G e r m a n y )