Degradation of Azlocillin in Human Serum

Zbl. Bakt. Hyg. A 260, 254-259 (1985)

Degradation of Azlocillin in Human Serum W. SCHONFELD,]. KNOLLER, K. D. BREMM, and W. KONIG Med. Mikrobiologie und Immunologie, Arbeitsgruppe Infektabwehrmechanismen, RuhrUniversitat Bochum, 4630 Bochum, Federal Republic of Germany

With 3 Figures· Received January 15, 1985 . Accepted March 1, 1985

Summary We developed a rapid and precise high performance liquid chromatographic method (HPLC) for the determination of azlocillin and its metabolites penicilloate and penilloate in serum and tissue as well as in vivo as in vitro. The linear relationship (r > 0.99) of determination ranged between 0.05 and 10 ug. No interference with other serum components was observed. The metabolism of azlocillin in vitro was analysed in serum. Within 24 h the amount of penicilloate increased from 1.5 % up to 18 % and from penilloate up to 1 % respectively. Buffer controls revealed a significant lower metabolism (4.7 % penicilloate, 0.9 % penilloate). These data suggest that the degradation of azlocillin does not only depend on bacterial ~-lactamase activity but is influenced by enzymatic activities within serum and human tissue.

Zusammenfassung Die Anwendung der Hochleistungsfltissigkeit-Chromatographie errnoglichr einen schnellen und sehr empfindlichen Nachweis des Acylureidopenicillins Azlocillin und seiner Metabolite Penicilloat und Penilloat sowohl in vitro als auch in vivo (Nachweisgrenze 50 ng, Linearitat zwischen 0.05 und 10 [!g bei einem Korrelationskoeffizienten von r > 0.99). In einer in vitro Studie wurde tiber 24 Std. der Abbau von Azlocillin in Serum und Pufferlosung verfolgt. Im Serum verbleiben nach 24 Std. noch ca. 50 % der eingesetzten Menge (Puffer 94 %), der Penicilloatanteil steigt auf 18 % (Puffer 4.7 %), Penilloat erreicht ca. 1 % (Puffer < 1 %). 31 % der eingesetzten Azlocillinmenge werden im Serum tiber einen nicht bekannten Abbauweg metabolisiert.

Introduction The analysis of antibiotic concentrations by microbiological and conventional biochemical techniques is often time consuming and laborious. A drug monitoring is only possible to a limited degree. Radioimmunoassays (RIA) and enzyme-linked-immunosorbent-assays (ELISA) are cost effective, easily to be performed but do not detect the metabolites due to the antibody specificity. Therefore the above described methods

Degradation of Azlocillin

a

255

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AZLOCILLIN

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N O=< N

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CH3 (H3 (OOH

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PENICILLOATE

N

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o=< N

0-I

CH-CO-HNU

~H CO

NH

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H3 c~

(DOH

PENILLOATE

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J

Fig. 1 do not pro ve whether low concentrations of the antibiotic in bod y fluids or tissues are caused by high metabolism of the compound or an impaired penetration into th e tissue compartment under study. Th e high performance liquid chromatography (HPLC) is able to solve this problem. Applications for nearly all ant ibiotics are published. The versatility and rapidity allows the drug-monitoring and the identificat ion of several substances in one chromatographic step, based on their chemical relationship and similar chro matographic behaviour; as an example serves the determination of different penicillins or metabolites (2, 4, 8, 9, 10, 11). In this report the method for the quantification of azlocillin (Securopen, Bayer AG, Leverkusen, FRG ) and its metabolites is described. Th is acylureidopenicillin with antibacterial activity against Gram-positiv e and Gram-negative bacteria especially against Pseudomonas aeruginosa was introduced in 1977. Numerous reports have dealt with the pharmacokinetic beha viour of azlocillin in various tissues (1, 3). Azlocillin is eliminated mainly without further alteration by kidney and in a lower amount by bile (1, 3). Moreover, azlocillin is metabolized by cleavage of the ~-I actam structure to penicilloare (Fig. 1); the subsequent decarboxylation leads to the penilloate (6). Few data are available about the degree of metabolism in vivo. Gau er al. reported that 6 % penicilloate appeared in urin e (4). Our own studies showed concentrations of 9 % penicilloate and 1 % penilloate in blood after infusion of 5 g azlocillin (9). N othing is known about the origin of these metabolites. It was the purpose of our experiment s to clari fy if the metabol ism takes place in blood itself.

256

W. Schonfeld,

J. Kneller,

K. D. Bremrn, and W. Konig

Materials and Methods Azlocillin kinetics: Sera of ten healthy donors and five buffer controls (phosphate buffer, pH 7.4) were used. The sera and buffer controls were incubated under sterile conditions at 37°C with azlocillin (400 ug/ml, gift from Bayer AG, Leverkusen, FRG). Aliquots of the samples were analysed before incubation with azlocillin (e.g. serum alone) directly after addition of azlocillin (zero value) and after 15, 30, 60, 90 min, 2, 4, 8, 12, 18 and 24 h of incubation. In preliminary experiments sterile urine was incubated with azlocillin (20 mg/ ml) for 24 h at 3rc. The determination of azlocillin and its metabolites by reversed phase HPLC was carried out by the following procedure. In brief: technical equipment- Constametric IIIG, Spectromonitor D, processing and integration unit CCM, automatic sample injector ASI 150 (LDC Milton Roy, Hasselroth). As solvent served acetonitrile/phosphate buffer (16: 84 v/v, pH 5.0); the phosphate buffer consists of 0.875 g KH 2P0 4 and 8.775 g K2HP0 4 • The flow rate was 1 ml/min with an injection volume of 20 ~I of a 1 : 2 diluted and filter (0.22 urn) cleaned sample. The column (4 X 200 mm) was filled with Nucleosil C18, 5 urn particle size (Macherey and Nagel, Duren). The run time amounted to 20 min with a detection wavelength of 220 nm at 0.05 A.U.F.S. (absorption units full scale). The identification of azlocillin and its metabolites is assessed by the determination of the retention time and the comparison with standards (Bayer AG, Leverkusen, FRG). The area integration of the absorption peaks allows the quantification of the substances (Fig. 2).

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Degradation of Azlocillin

257

Result s Our data present evidence for degradation of azlocillin in human serum. The here described method is a practicable tool for a rapid in vivo and in vitro determination of azlocillin and its metabolites in blood and tissue with a detection limit as low as 50 ng. The linearity of determination ranges betwe en 0.05 and 10 ug) correlation coefficient r > 0.99, precision + /- 5 %) . As is apparent from Fig. 2a clear cut sepa ration of standards consisting of azlocillin, penicilloate and penilloate is demonstrated. Incubation of azlocillin with serum over 24 h leads to a profound increase in penicilloate. The experiments were performed using fresh sera of ten different donors. Azlocillin at a concentration of 400 ug/ml was incubated up to 24 h. At various time intervals the amount of azlocillin and the genera ted penicilloate and penilloate was immediately

Table 1. Metabolism of azlocillin in serum and buffer (means ± S. D. of five replicates) time

o min

serum concentrations (ug/rnl) 120 min 240 min 12 h

60 min

azlocillin 394±44 penicilloate 6± 1 penilloate 1.7± 0 deficit amount 0

395± 59 7± 1 3.4±0.5 0

356±33 20±1l HO.9 14

349±40 20±11 2±0.6 29

buffer concentrations (ug/rnl) 24 h

time

o min

azlocillin penicilloate penilloate

400± 8 6±0.5 HO

372±10 18± 1.4 3.6± 0.5

ug/ mt

t-t---t-t--H -i--t-

h.f.-.. t

AZLOClLLIN 100

PENIClLLOATE

d-I/

10

t--f-1- 1

PENILLOATE

f- i-+o

L f/yt

- 1/ 1

15 30 60 90 min 2

Fig. 3

j/]t1

4

8 121824h

280±30 38±1 7 3± 1 82

24 h 193±23 7D± 36

5± 1 134

258

W. Schonfeld, J. Kneller, K. D. Bremm, and W. Konig

analysed by reversed pha se HPLC. Azlocillin is metab olized by 50 % within the rime range of 24 h. Penicilloate reaches about 18 % and penillo ate about 1.2 % of the azlocillin concentration. If one calculates from these data the tot al sum of all three compounds 134 ug (33 %) of substance s are lacking to obtain the original concentration of 400 ug, which was applied at the beginning of the incub ation. These data suggest that additional metabolites are generated which are not determined under th e chro matographic condi tions (see Ma terials and Methods). Table 1 furt her clarifies the results obtained fro m th e experiment demonstrated in Fig. 3. As can be seen with the higher metabolism of azlocillin in serum the amount which is not detected ranges from o ug to 134 ug after 60 min and 24 h of incubation respectively. In buffer azlo cillin behave s rather sta ble as is shown over a time range of 24 h of incubation (Table 1). The percentage of penicilloate rises from 1.5 % at 0 min up to 4.5 % after 24 hand penilloate from 0.5 up to 0.9 % respectively. Discussion While the pharmacokinetic behaviour of azlocillin has been often studied in the last years few data are available with regard to the metabolism of azlocillin. By HPLC analysis a quantification of azlocillin as well as the metabol ites penicilloate and penilloate were achieved in one chromatographic step . The cleavage of the ~ -l a ctam structure by bacteria has been widely examined (6, 4). In contrast the generation of metabolites by non-bacterial mechan isms has not been described . We recently described that under in vivo conditions 9 % penicilloate is generated (8); these data are also confirmed for the in vitro conditions; dur ing the first hours, wh ich are impo rtant for therapy, azlocillin behaves rather stable; further incubation leads to a remark able metabolism via penicilloate in serum which contrasts to the stabil ity in buffer. In ad dition these data suggest different ways of metabolism. At this point it is not possible to decide whether the resulting products are derived from azlocillin or penicilloate or penilloate. The fact that a rather large amo unt of the original azlocillin is nor detected under the described condition s may be due ro the formation of small, polar products, which elute rap idly from the column or due to the formation of a non-absorb ing substance. Recent stu dies show also degradation of azlocillin in urine leading ro penicilloate and penilloate (data not shown here). The role of pH and osmola rity as to the stability of azlocillin will be analysed in future studies. There is evidence that azlocillin is destroyed in biolo gical fluids and tissue (8). This effect is however not easily seen in vivo which is probably due to th e fast elimination via kidney and the bile. On the other hand one also has to consider a destruction of azlocillin during the storage of samples even at low temp eratures. This has been shown when azlocillin was stored in buffer solution at -20 °C (10). Further studies are carried out to characterize the still unknown metabolites.

References 1. Bergan, T.: Review of the pharmacokinetics and dose dependency of azlocillin in normal subjects and patients with renal insufficiency. J. Anrimicrob. Chemother. 11, Supp/. B (1983) 101-114 2. Das Gupta, V.: Stability of mezlocillin sodium as determined by high-performance liquid chromatography. J. Pharm. Sci. 72 (1983) 1479-1481

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259

3. Diiben, W., E. Helu/ing, D. Forster, I. Putter und D . Tettenborn: Die biliare Exception von Azlocillin. Fort sch. Med. 97 (1979) 419-421 4. Gau, W. und F. A . Horster: Hochdruckfliissigkeitschromatograph ische Anal yse von Azlocillin und seinem Penicilloat im Urin. Arnzeimitt.-For sch. 29 (1979) 1941-1943 5. Hamilton-Miller, J. M. T. and ]. T. Smith: Beta-Lactamases. Academic Press, Lond on (1979) 6. Jacobs, J. Y., D . M. Livermore, and K. W. M. Davy: Pseudomonas aeruginosa Blactamase as a defence against azlocillin, mezlocillin and piperacillin. J. Ant imicrob. Chemother. 14 (1984) 221- 229 7. Konig, H. B., K. G. Metzer, H. A. O ffe und W. Schrock: Azloeillin. Ein neues Penicillin aus der Acylureidoreihe. Europ .]. Med . Chern. - Chim. Ther. 17 (1981) 59-63 8. Nilsson-Ehle, I.: High performance liquid chromatograph y for analyses of ant ibiot ics in biological fluids.]. Liq. Chro matogr. 6 (1983) 25 1-293 9. Schonfeld, W ., J. Kneller, K. D. Bremm, R. Ritscher und W. Konig: Antibiotikabestimmung durch HPLC am Beispiel des Azlocillins. In: Fort schritte der antimikrobiellen und antineoplastischen Chemother apie. Futuramed Verlag, Miinch en (1985) 10. Thijssen, H. H. W.: Analysis of isoxazolyl penicillins and their metabolites in bod y fluids by high-performance liquid chromatography. J. Chromatogr. 183 (1980) 339-345 11. Weber, A., K. E. Oph eim, K. Wong, and A. L. Smith: High-pressure liquid chromatography quantification of azlocillin. Antimicrob. Agents Chemother. 24 (1983) 750-753 Dr. med. W. Schonfeld, Prof. Dr. med. W. Konig, Med . Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr-Uni versitat Bochum, Universitatsstr, 150 , 0-463 0 Bochum