F r o m Lehrstuhl für Pharmakologie
1und Rechenzentrum
2der U n i v e r s i t ä t Regensburg and Urologische K l i n i k
3, 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
By F. Rees
1, K . G . Naber
3, B. Bartoschik-Wich
3, P. S t o c k m a n n
3, G . P. M e y e r
2, and H . Grobecker
1Summary: In plasma and urine of 10 healthy volunteers
after intravenous administration of 4 g mezlocillin and pi- peracillin, respectively, the parent compounds as well as degradation products were assayed by high-performance li-
quid chromatography. Ioxitalamic acid, a renal contrast me-
dium, 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 piper- acillin 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 anti- biotics.
Zusammenfassung: Hochdruckflüssigkeitschromatographi-
sche Bestimmung von Mezlocillin, Piperacillin, ihrer Abbau- produkte und von loxitalaminsäure im Plasma und Urin ge- sunder Probanden
Nach intravenöser Applikation von je 4 g Mezlocillin bzw.
Piperacillin wurden mit Hilfe der Hochdruckflüssigkeits-
chromatograpihie die Muttersubstanzen sowie Abbaupro- dukte 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 loxi- talaminsäure, ein Kontrastmittel, infundiert. Als einziger Metabolit vom Mezlocillin wurden geringe Mengen der entsprechendem Penicilloinsäure gefunden. Im Falle von Pi- peracillin wuirde ein weiteres Abbauprodukt beobachtet.
Etwa die Hälfte der gegebenen Antibiotika-Dosen wurde un- verändert im Urin wiedergefunden, zusätzlich noch 5-10%
in Form von Abbauprodukten. Bei den pharmakokineti- schen Parametern beider Penicilline wurden keine signifi- kanten Untersschiede festgestellt.
Key words: Acylureidopenicillins • Diagnostics • Ioxitalamic acid, clinical pharmacokinetics • Mezlocillin, clinical
pharmacokinetics • Piperacillin, clinical pharmacokinetics
1. Introduction
M e z l o c i l l i n (6-[D-2-(2-oxo-3-mesylimidazolidine-1 -carbox- amido)-2-phenylacetamido]penicillanic acid) and piperacil- l i n (6-[D(-)-a-(4-ethyl-2,3-dioxo-l-piperazinyl-carbonyl-ami- no)-2-phenylacetamido] penicillanic acid) are acylureido penicillins with broad antibacterial activity and with only slight differences between their structures (Scheme 1). Peni- cillins are i n general partially metabolized, m a i n l y to inactive penicilloic acids [1]. T h i s also was shown for mezlo- c i l l i n , but i n vitro degradation o f m e z l o c i l l i n in plasma specimens may have influenced some results, leading to false high concentrations o f m e z l o c i l l i n 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].
and 188 c m (miedian 174 cm). T h e health o f the volunteers was established fromi their medical history, p h y s i c a l e x a m i n a t i o n and l a - boratory 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 p e n i c i l l i n s o r contrast m e d i a . Pregnancy was ex- cluded by the retgular use o f contraceptives.
2.2. Administration of antibiotics
After giving infformed written consent the subjects received 4 g o f m e z l o c i l l i n and after 6 weeks 4 g o f p i p e r a c i l l i n . C o n c u r r e n t l y , the subjects receivecd 5 m l T e l e b r i x 300®, equivalent to 2.4 g i o x i t a l a m i c acid, as an inteirnal standard for c o m p a r i s o n o f the two antibiotics.
T h e injection o f this contrast m e d i u m allows to measure the renal function as glormerular filtration rate, and to c o n t r o l the collection o f the 24-h urine. T h e antibiotics and i o x i t a l a m i c 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 - C H - C 0 - N H v * ^ S
Ph i rs
R =
R = • - 0
• n 2 L
R - C H - C O - N H . S
C00H OH
m e z . penicilloic acid
N N- Et1 : p i p e r a c i l l i n pip. penicilloic acid 0 o
Ho-U (
> - H N N- Et- H90
+ H 2
\ /
- C O :
R - C H- C 0- N HX Ph
C:OOH C00H
mez penilloic acid
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 pharmaco- kinetics of mezlocillin and piperacillin after intravenous i n - fusion o f 4 g over 30 m i n , a n d to quantify their probable main metabolites, the corresponding penicilloic acids. F o r analysis a liquid chromatographic method was chosen i n - stead o f bioassay, because these ring-open p e n i c i l l i n deriva- tives are not antibacterially active. Care was taken w i t h the sampling and storage o f plasma samples because o f in vitro instability o f mezlocillin and other penicillins i n body fluids [3].
2. Material and methods 2.1. Subjects
T h e study included 10 healthy subjects (5 male, 5 female, aged be- tween 22 and 34 years, median 29 years). T h e i r weights ranged be- tween 50 and 86 kg (median 66 kg), and their heights between 154
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 o f infusion and at 10, 20, 30, 4:5, 6 0 , 9 0 m i n , 2, 3, 4, 6 and 8 hours thereafter. B l o o d samples were ccentrifuged at 4 °C w i t h i n 30 m i n and the plasma was stored at - 7 0 ° C u n t i l analysis. U r i n e samples were collected before the start o f the experiments and d u r i n g 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 o f a l l urine samples were measured, and the specimens were stored at - 7 0 °C. T h e excretion o f creatinine i m the fractionated urine collections over 24 h was as- sayed by the auitoanalyser modification o f the method o f JafTe (4).
2.4. Chromatographic assay 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 w i i t h its penicilloic acid and i o x i t a l a m i c 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 rrueg- l u m i n e ioxitalamate ( T e l e b r i x 3O0®; manufacturer: B y k G u l d e n , K o n s t a n z , F R Germainy)) was used.. A c e t o n i t r i l e ( H P L C grade S) was purchased from Z i n s s e r , F r a n k f u r t / M a i n ( F R G e r m a n y ) , tetrabutyl- a 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 a n t i b i o t i c s , their metabolites and o f i o x i t a l - amic acid were p r e p a r e d in waiter to yield finail concentrations of 1 m g / m l , and stored i n a l i q u o t s 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 m e z : l o c i l l i n and p i p e r a c i l l i n a n d 2 0 / i g / m l for the p e n i c i l l o i c acids o f b o t h p e n i c i l l i n s . F o r c h e c k i n g t h e linearity o f the assay dilutions o f 1 0 O - 0 . 7 8 ^ g / ' m l 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 pre- pared. L a c k o f sufficient material d i d not a l l o w to prepare a stand- ard 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 m i n o r m o d i f i c a t i o n s . In brief, 200 p\ plasrma were buffered with 200 ß\ 50 m m o l / 1 s o d i u m phosphate ( p H 6.(3) and deprotein- ized with 400 ß\ a c e t o n i t r i l e . T h e latter was than extracted into 2 m l dichloromethane, amd 1 0 - 2 0 iA o f the aqueous phase, c o n t a i n i n g the penicillins and t h e i r metabolites, were injected into the C h r o m a - tograph. U r i n e was centrifuged a n d diluted t e n f o l d 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 au- tomatic injector W I S P 7 1 0 B (fitted w i t h a c o o l i n g kit, in o r d e r to maintain 8 °C for the samples), a R C S S c o m p r e s s i o n 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 4 4 1 , 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 A s s o c . , 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 . t h e resulting back- pressure 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 d e t e r m i n a t i o n o f m e z l o c i l l i n and at 2129 n m ( C d - l a m p ) 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 so- d i u m phosphate buffer ( p H 6.8), 240 m l acetoniitrile and 150 mg t e t r a b u t y l a m m o n i u m hydrogen sulfate. T h e appairent p H was ad- justed 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 u s i n g 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 p i p e r a c i l l i n assay 1 g tetrabutyl- a m m o n i u m hydrogen sulfate was used and the p H adjusted to 6.5.
F o r 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 w i t h 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 m a i n t a i n e d at 1.0 m i / m i n , the back-pressure was 11000 k P a . T h e m o b i l e phase was a mixture o f 9 1 0 m l water, 90 m l a c e t o n i t r i l e , 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. T h e 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 ( H g - l a m p ) , the retention t i m e olf i o x i t a l a m i c a c i d was about 4.5 m i n .
2.4.4. Pharmacokinetic analysis
Plasma level data were analysed b y the open, t w o - c o m p a r t m e n t 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 r e l a - tive least-squares regression analysis. A F o r t r a n p r o g r a m was used in the computation. T h e basic equation o f the m a t h e m a t i c a l m o d e l was
Cj> = A • e-*1 + B • ,-ßt
where Op represents the plasma concentrations att time t after the dose. 2 and ß ( m i m1) are h y b r i d constants o f the fast and the s l o w disposition process, respectively, and A a n d B (mig/1) are the zero- time intercepts of the t w o components o f the biex.ponential curves.
T h e calculated p h a r m a c o k i n e t i c constants were corrected for infu- sion t me [6]. A n u m b e r o f parameters have been calculated, espe- cially following: area under the plasma concentiration-time curve (AUG).«,): apparent steady-state v o l u m e o f d i s t r i b u t i o n (V$s), total body :learance ( C ll 0t ) , a n d t e r m i n a l plasma half-life* (U/iß)-
£ 0 k 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 s o l u t i o n 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 a n d 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 m i x t u r e o f epimers A and B), (2) a p l a s m a b l a n k a n d (3) p l a s m a 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 (*) m a r k s a i unidentified substance that appears in treated p l a s m a o r aqueous samples, a i d disappears w i t h i n 2 days at r o o m temperature (see c h r o m a t o g r a m 2). Concentrations:
M E Z = 15 / / g / m l . M P C = 2.5 ^ g / m l . A U = absorption units.
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 : C o l u m n : H I B A R L i C h r o s p h e r R P - 1 8 5 ^ m ; (125 x 4 m m I . D . ) ; m o b i l e 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 - l i n and its degradation products, m e z l o c i l l i n pen c i l l o i c and p e n i l l o i c acid, and o f extracted human plasma are depicted.
Each m e z l o c i l l i n metabolite elutes as two distinct peaks be- cause of epimerization at the carbon atom i n position 5 (see Scheme 1) [7]. T h e earlier eluting isomers A are the m i n o r components in freshly prepared aqueous solutions, but be- come d o m i n a n t when standing at r o o m temperature for m a n y hours as it was also described for a m o x y c i l l i n p e n i c i l - loic a c i d [8]. Isocratic separation o f a l l compounds w i t h i n 10 minutes, as seen i n F i g . 1, was enabled by application o f re- versed phase ion pair chromatography. F o r L i C h r o s p h e r R P 18 silica a good c o m p r o m i s e 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 tetra- b u t y l a m m o n i u m hydrogensulfate in the m o b i l e phase and p H 6.2. F o r N o v a p a k C-18 silica a lower content o f tetra- b 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 p l a s m a and urine, we found m e z l o c i l l i n penicilloic a c i d o n l y . Since it has two c a r b o x y l i c groups, the retention time is more sensitive to changes i n concentration of tetrabutyl- a m m o n i u m salt and p H than that of m e z l o c i l l i n hself.
F i g . 2 shows chromatograms of a standard mixture o f piper- a c i l l i n and p i p e r a c i l l i n p e n i c i l l o i c acid, prepared by incuba- t i o n o f p i p e r a c i l l i n w i t h ^-lactamase, and o f plasma samples o f 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 , p i p e r a c i l l i n p e n i c i l l o i c acid elutes as two separated epimers, and is hardly to deter- m i n e i n 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. T h e areas of the two unidentified metabolites o f p i -
25*» nm
0 . 0 0 2 A U
P P C A B
2 2 9 nm
QJ «-
^ 0
r
U min 8
CD 1
'.E 0 4 min 8 - 0
P I P
X A B
I IUI
PPC A Bj ~ « * v
X A B
4 min 8 :e 0
Ii
i* min 8Fig. 2: C h r o m a t o g r a m s o f ( 1 ) an aqueous standard s o l u t i o n o f p i p e r a c i l l i n ( P I P , 250 ng) a n d p i p e r a c i l l i n p e n i c i l l o i c a c i d ( P P C , c a . 200 ng m i x t u r e o f epimers A a n d B ; the asterisk m a r k s an i m p u r i t y , p r e s u m a b l y a hydrolysis product o f piperacillin), (2) a plasma b l a n k , (3) p l a s m a o f a v o l u n t e e r 1 h a n d ( 4 ) 4 h after the e n d o f an intravenous infusion (30 m i n ) o f 4 g p i p e r a c i l l i n . X marks two unidentified peaks ( A a n d B), probably a m i x t u r e o f e p i m e r s . concentrations: (3) P I P = 47 ßg/ml P P C = 2,3 / i g / m l , X = 9,8 # g / m l w h e n 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 a n d m e t h - ods. 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 m e z l o c i l l i n penicilloic acid 1 1 0 0 . 8 ± 2 . 8 % (concentration 42 / i g / m l ; n = 9), for piper- acillim 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 p e n i c i l l o i c a c i d 91 to 93% (concentration 20 / i g / m . l ; n = 3). T h e recovery from urine was not checked and set 100%, as the o n l y sample treatment step was dilution of urine with buffer.
S o m e plasma specimens o f the m e z l o c i l l i n study were deter- m 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 re- sults are shown i n T a b l e 1.
3.3. Pharmacokinetics
Fig. .3 and 5 show the mean ( ± S D ) plasma concentrations and the cumulative u r i n a r y excretion o f mezlocillin and its ring-open metabolite, F i g . 4 a n d 6 the respective data of p i - p e r a c i l l i n . In addition, the plasma concentrations of ioxita- l 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 - l i n a d d e d : 2 5 , 5, 0.5 / / g / m l . )
D a y P i p e r a c i l l i n (//g/ml)
1 25.2 5.69 0.60
2 26.0 5.85 0.69
3 25.3 5.29 0.62
4 26.1 5.96 0.69
5 26.2 5.74 0.70
M e a n 25.8 5.71 0.66
S D (%) 0.5(1.8%) 0.25 (4.5%) 0.05 (7.0%)
ently neither m e z l o c i l l i n nor piperacillin is metabolized in vivo to a greater extent. These findings are also illustrated i n T a b l e 2. In both studies we found nearly identical values for ioxitalamic acid in plasma, and in the beginning also for m e z l o c i l l i n and p i p e r a c i l l i n . W i t h the time elapsed, the concentrations of m e z l o c i l l i n remained slightly higher than those of p i p e r a c i l l i n .
T h e pharmacokinetic parameters of m e z l o c i l l i n , piperacil- l i n , and o f i o x i t a l a m i c acid are summarized in Table 3. A l l substances exhibit distribution volumes nearly 20% o f body weight w h i c h agrees good with the extracellular space. L i k e iotalamic acid [9], i o x i t a l a m i c acid shows the same plasma clearance as i n u l i n (ca. 120 m l / m i n ) , whereas those of mez- l o c i l l i n and p i p e r a c i l l i n are about 250 m l / m i n . O n the other hand, the half-life o f ioxitalamic acid i n plasma is about 2 h, whereas the half-lives o f both antibiotics are 1 h. T h e u r i - nary recovery of i o x i t a l a m i c acid within 24 h was 90% in each study so that complete 24-h urine collection can be as- sumed. A l s o the excretion of creatinine was normal i n all subjects. A b o u t 50% o f the antibiotic doses given were reco- vered in the urine as active parent compounds, and 9% as metabolites i n the case of piperacillin and 4% in the case o f m e z l o c i l l i n .
4. Discussion
4.1. Chromatographic assay
Besides the traditional bioassay, m e z l o c i l l i n and piperacillin have been also determined in biological fluids by reversed phase H P L C [10-15]. But in a l l these cases the parent c o m - pounds only were assayed, as the ring-open metabolites are m u c h more polar and elute with the front. U s i n g gradient elution technique m e z l o c i l l i n [16], a z l o c i l l i n [17], a p a l c i l l i n [18], and the respective penicilloic acids could be deter- mined simultaneously i n serum and urine. Unfortunately, all assays are considerably time consuming. In the present assay incorporation o f ion-pair chromatography enabled iso-
T a b l e 2 : M e a n ( ± S D ) c o n c e n t r a t i o n s 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. a n d 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 p i p e r a c i l l i n study: I T X m e z = i o x i t a l a m i c a c i d in the m e z l o c i l l i n s t u d y ; 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 .
T i m e I T X p i p (/ig/ml)
I T X m e z Gwg/ml)
I T X m e z I T X p i p
M E Z (jug/ml)
P I P (A*g/ml)
M E Z 100 (%) P I P
- 1 5 m i n1' 141 ± 2 7 134 ± 4 0 95.0 200 ± 58 198 ± 4 2 110.0
0 m i n2» 194 ± 5 1 187 ± 4 5 96.4 271 ± 6 1 272 ± 7 5 99.6
10 m i n 142 ± 1 8 144 ± 3 5 101.4 198 ± 4 6 187 ± 3 3 105.9
20 m i n 126 ± 1 6 121 ± 2 7 104.3 158 ± 3 9 148 ± 2 6 106.8
30 m i n 110 ± 1 5 108 ± 2 4 98.2 129 ± 3 4 118 ± 2 6 109.3
45 m i n 94.9 ± 15.9 9 1 . 3 ± 1 7 . 9 96.2 9 7 . 7 ± 2 5 . 8 8 7 . 7 ± 2 2 . 5 111.4
60 m i n 79.5 rt 11.5 7 8 . 5 ± 16.0 98.7 7 3 . 0 ± 2 1 . 8 6 5 . 2 ± 1 4 . 5 112.0
90 m i n 6 2 . 5 ± 9.9 6 I . 0 ± 1 2 . 2 97.6 45.1 ± 1 6 . 5 3 9 . 6 ± 1 1 . 2 113.9
2 h 49.3 ± 9.9 4 9 . 2 ± 10.8 99.8 2 9 . 5 ± 1 3 . 4 24.1 ± 8.4 122.4
3 h 3 2 . 0 ± 8.2 3 2 . 0 ± 7.5 100.0 1 2 . 5 ± 4.7 I 1 . 5 ± 5.8 108.7
4 h 22.1 ± 6.0 21.1 ± 6.9 95.5 6.0 ± 2.4 5 . 5 ± 3.1 109.1
6 h 1 0 . 4 ± 3.0 9 . 6 ± 3.9 92.3 1 . 6 ± 0.8 1 . 5 ± 0.6 106.7
8 h 5.1 ± 2.3 5 . 0 ± 2.4 98.0 0.6 ± 0.4 0 . 6 ± 0.2 100.0
M e a n ± S D 9 8 . 0 ± 3 . 1 1 0 8 . 2 ± 6 . 2
1' 15 m i n after the start of i n f u s i o n (the a n t i b i o t i c s were infused at a constant rate over 30 m i n ) .
2 ) A t the end o f 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 ± S D ) 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 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 a c i d (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 a c i d i n the m e z l o c i l l i n test; I T X p i p = i o x i t a - l a m i c acid in the p i p e r a c i l l i n 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 concentra- tion-time c u r v e ; C l t = total body c l e a r a n c e : iß - t e r m i n a l plasma half-life; Vss% b.w. = Vss i n percentage o f body weight; C l t 70 kg = C l t 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.
Vss Vss% b.w. A U C Clt C lt7 0 kg Uo-24 h])
(1) (%) (mg/1 h) ( m l / m i n ) ( m l / m i n / 7 0 kg) (min) (% o f dose)
I T X m e z 15.5 ± 3.2 2 3 . 5 ± 3 . 6 3 4 9 ± 7 1 1 1 8 ± 2 2 1 2 7 ± 2 7 106 ± 1 5 93.1 ± 7.1
I T X p i p 15 + ? 6 2 3 . 3 ± 4 . 5 359 ± 5 9 1 I 4 ± 1 8 1 2 3 ± 2 7 107 ± 1 4 8 7 . 2 ± 1 1 . 4
M E Z 14.3 + 3.8 21.8 + 5.1 303 ± 7 6 231 ± 5 0 247 ± 5 2 6 3 . 9 ± 1 1 . 0 4 9 . 6 ± 4.72)
P I P 1 4 . 5 ± 2 . 7 2 2 . 2 ± 4 . 7 2 8 2 ± 6 l 246 ± 5 0 262 ± 5 4 6 6 . 4 ± 1 3 . 0 4 9 . 6 ± 3.83)
') 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)).
2> 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 a c i d .
3» In a d d i t i o n : 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 a c i d , a n d 5 . 0 ± 1.5% as c o m p o u n d X .
cratic determination of m e z l o c i l l i n , p i p e r a c i l l i n , and their probable m a i n 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). M o r e o v e r , it seems doubtful whether the measured concentrations o f the penicilloic acids are caused by enzy- matic metabolization, or m a i n l y by mere chemical hydroly- sis. T h e instability of these and other penicillins i n standing plasma specimens was several times reported, and even at - 1 8 °C 20% degradation o f m e z l o c i l l i n in plasma w i t h i n 6 weeks was observed, whereby half of it was found in form o f m e z l o c i l l i n penicilloic acid [3]. Therefore, the detection o f large amounts o f m e z l o c i l l i n penicilloic acid i n any body fluid specimen may indicate rather in v i t r o degradation o f the parent c o m p o u n d than in vivo metabolism. In such cases, false low concentrations o f m e z l o c i l l i n are measured, and determination o f degradation products can prove it (cf.
[3]). In accordance with results examined by G a u and För- ster [16] we d i d not find m e z l o c i l l i n p e n i l l o i c acid in plas- m a and urine, and indeed, the detection o f p e n i l l o i c acid in body fluids [19] may have been an artefact o f the analytical procedure [3].
E v e n quantitative disappearance o f p i p e r a c i l l i n from plasma specimens was stated when stored for several weeks at - 2 0 °C ([10]; unfortunately, though H P L C was used, no at- tempt 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 o f p i p e r a c i l l i n should be guar- anteed. M o r e o v e r , we found only small amounts o f piper- a c i l l i n p e n i c i l l o i c acid, the supposed m a i n degradation pro- duct i n stored plasma. But beyond that, two unidentified sig- nals appeared i n 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 i n Fig. 1, w h i c h 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 o f piperacillin and p i p e r a c i l l i n p e n i c i l l o i c 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 o f the p e n i c i l l o i c acids of m e z l o c i l l i n and a p a l c i l l i n ([18]; unfortunately, p i p e r a c i l - l i n p e n i c i l l o i c acid was hardly to quantify in plasma, a l l the more since the isomer A was often covered by an endoge- nous compound). T h i r d , the same splitting o f the d i o x o p i - perazine ring was foundi i n the case o f cefoperazone [20].
T h e Baternan-function-like concentration-time course o f c o m p o u n d X i n plasma (Fig. 4) demonstrates it to be formed in v i v o , presumably as a metabolite o f p i p e r a c i l l i n p e n i c i l - loic acid. H o w e v e r , the identity o f 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 i n a randomised cross-over fash- i o n but the analysis o f the pharmacokinetic parameters o f ioxitalamic acid i n both tests (Table 3) revealed nearly iden- tical mean values so that s i m i l a r conditions can be assumed to exist for both antibiotics m e z l o c i l l i n and p i p e r a c i l l i n . A l l their mean pharmacokinetic parameters differed not more than 10% from each other which is w i t h i n the analytical er-
ror, and in accordance with published data [21, 22]. W e ob- served also the same urinary recovery o f both antibiotics, about half o f the dose given, which was to be expected for m e z l o c i l l i n only [3, 16, 21]. Tjandramaga [22] found 80% o f p i p e r a c i l l i n i n 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 o f 4 0 - 7 0 % renal excretion of p i p e r a c i l l i n is also reported (cf. [23, 24]). So, for piperacillin a very varia- ble range o f renal excretion is found. O u r data are at its lower margin, but in good agreement with the renal excre- tion o f the other acylureido penicillins, m e z l o c i l l i n [3. 16, 21] and a z l o c i l l i n [17], which exhibit very s i m i l a r c h e m i c a l structures.
5. References
[1] B a x . R . P.. W h i t e . L . O . . B y w a l e r . M . . Reeves. D . S., N . Engl. J . M e d . 304, 734 (1981) - [2] See rcf. [22]. discussion - [3] Kees, F., Naber. K . G . , D o m i n i a k . P., S t o c k m a n n . P.. M e y e r . G . P., A d a m . D . , G r o b e c k e r . H . , Fortschr. A n t i m i c r o b . A n t i n e o p l a s t . C h e m o - ther., in press - [4] D i G i o r g i o . J . , i n : H e n r y . J . R . , C a n n o n . D . C , W i n k e l m a n , J. W . (eds.), C l i n i c a l Chemistry. P r i n c i p l e s and T e c h n i c s , 2nd ed., p. 541. H a r p e r and R o w . P u b L Inc.. Hagerstown (1974) - [5] Kees. F . , G r o b e c k e r . HL, Naber. K . G . . J . C h r o m a t o g r . 305, 363 ( 1 9 8 4 ) - [6] G i b a l d i , M . , Perrier. D . . i n : Swarbrick, J . (ed.), Drugs and the Pharmaceutical Sciences. V o l . 1. Pharmacokinetics, p. 69, M a r c e l D e k k e r , Inc.. N e w Y o r k (1975) - [7] K ö n i g . H . B..
Metzger, K . G . , Offe, H . A . , S c h r ö c k , W . , A r z n e i m . - F o r s c h ./ D r u e , Res. 33 (I), 88 (1983) - [8] F o n g , G . W . K . , J o h n s o n . R . N . . K h o , B.
T . , J . Chromatogr. 6, 2513 (1983) - [9] Naber, K . G . . Kees, F . , A d a m , D . , M e y e r , G . P., Johnson, L . C , Letzel. H . . G r o b e c k e r , H . , Zschr. A n t i m i c r o b . Antineoplast. Chemother. 2, 161 (1984) - [10]
Jung, D . , M a h a j a n , N . K . , C l i n . C h e m . 30, 122 (1984) - [11] Bris- son, A . M . , F o u r t i l l a n , J . B . . A n t i m i c r o b . Agents Chemother. 21, 664 ( 1 9 8 2 ) - [12] Meulemans, A . . M ö h l e r , J . , Decazes, J . M . , Dous- set, I., M o d a i , A . , J . L i q u i d C h r o m a t o g r . 6, 575 (1983) - [13] A r a - v i n d . M . K . , M i c e l l i , J. N . . K a u f f m a n , R. E . , J. C h r o m a t o g r . 233, 423 ( 1 9 8 2 ) - [14] Hildebrandt, R . , G u n d e r t - R e m v , U . , J . C h r o m a - togr. 228, 409 (1982) - [15] Fasching, C . E . , Peterson. L . R . . J. L i - q u i d Chromatogr. 6, 2513 (1983) - [16] G a u . W . , F ö r s t e r , D . , 20th International Congress on A n t i m i c r o b i a l Agents and C h e m o t h e r a p y , N e w Orleans, U S A , 2 2 - 2 4 Sept., Abstract 2 5 2 : A m e r . Soc. M i c r o - b i o l . (1980) - [17] G a u , W . , Horster. F. A . , A r z n e i m . - F o r s c h . D r u g Res. 29 (II), 1941 ( 1 9 7 9 ) - [18] Borner, K . , i n : L o d e , H . (ed.). L u - mota ( A p a l c i l l i n ) - ein neues A n t i b i o t i k u m , 2. A u f l . , p. 55, P m i - pharm-and-medical-information-Verlags G m b H . F r a n k f u r t / M a i n (1983) - [19] Graber. H . . A r r , M . , C s i b a . A . , L u d w i g . E . , Magyar, T . , Int. J . C l i n . Pharmacol. T h e r . T o x i c o l . 19, 539 (1981) - [20]
K e m m e r i c h , B . , Lode, H . , Borner, K . , Belmega, D . , Jendroschek, T . , K o e p p e , P., G o e r t z , G . , .1. A n t i m i c r o b . Chemother. 12, 27 (1983) - [21] Lode, H . , Koeppe, P., K e m m e r i c h , B . , Niestrath, I L T o m a s . W . , Belmega, D . , Jendroschek, H . . i n : Siegenthaler, W . . Weuta, H . (eds.), Berichte ü b e r das Internationale S y m p o s i o n A c v l u r e i d o - P e n i c i l l i n e , W i e n 1979, p. 45, Excerpta M e d i c a . A m s t e r d a m (1980) - [22] Tjandramaga, T . B . , i n : N o r d b r i n g , F. (ed.). P i p e r a c i l l i n , a p e n i c i l l i n for serious infections, p. 39. Excerpta M e d i c a . A m s t e r d a m (1983) . - [23] Baier, R . . R u p p e l . H . . Zelder. O . . Zehner. R . . A r z - neim. - F o r s c h . / D r u g Res. 31 (I), 857 (1981) - [24] L o d e . H . . Elvers, A . , K o e p p e . P.. Borner, K . . A n t i m i c r o b . Agents C h e m o t h e r . 25. 105 (1984)
Acknowledgements
W e wish to thank D r . A . H u b e r from Bayer A G . M u n i c h , for sam- ples o f m e z l o c i l l i n , 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 o f p i p e r a c i l l i n .
F o r the authors: Prof. D r . med. H . Grobecker, L e h r s t u h l für Phar- makologie 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 o f G e r m a n y )
