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Clavulanic Acid Decomposition Is Catalyzed by the Compound Itself and by Its Decomposition Products
NOTES Clavulanic Acid Decomposition Is Catalyzed by the Compound Itself and by Its Decomposition Products SIMONE BRETHAUER, MARTIN HELD, SVEN PANKE ETH Zurich, Bioprocess Laboratory, Institute of Process Engineering, Universitaetsstrasse 6, 8092 Zurich, Switzerland
Received 21 June 2007; revised 31 August 2007; accepted 14 September 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21225
ABSTRACT: The decomposition kinetics of the b-lactamase inhibitor clavulanic acid (CA) was investigated for CA concentrations between 2.5 and 20 g L1, which is assumed to represent a characteristic range for an industrial CA production process. For each initial concentration, first order kinetics plots could be obtained, however the kinetic constant increased from 3.8 103 to 8.6 103 h1 with increasing initial CA concentration, indicating that CA accelerates its own decomposition by general acid–base catalysis. Furthermore, the kinetic constant remained approximately constant during the reaction, suggesting that also the decomposition products of CA had to show similar catalytic activity. This was confirmed experimentally by increased CA decomposition rates when CA degradation products were added to the reaction. A kinetic model is proposed, which is able to reliably predict the observed pseudo first order rate constants. The presented results should be considered in any process where highly concentrated CA solutions are employed, for example, during final downstream processing or in industrial fermentations. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3451–3455, 2008
Keywords:
chemical stability; kinetics; anti-infectives; HPLC; mathematical model
INTRODUCTION Clavulanic acid (CA) (1) ((2R,3Z,5R)-3-(2-hydroxyethyliden)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptan-2carboxylic acid) is a fused bicylic b-lactam molecule produced by Streptomyces clavuligerus. CA is one of the active ingredients in Augmentin1 and acts as a potent inhibitor of b-lactamases produced by some b-lactam resistant pathogenic microorganism to enable efficient treatment of infectious diseases.1 The production as well as the downstream processing is accompanied by the compound’s degradation, initiated by hydrolysis, which is around 10 times faster than the one of penicillin G.2 Correspondence to: Sven Panke (Telephone: þ41-44-632-0413; Fax: þ41-44-632-19-93; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 3451–3455 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
Hydrolysis of CA under neutral or basic conditions proceeds via the reactive amino ketone (2) to pyrazine end-products (3–5) (Fig. 1),3,4 which are part of a complex product mixture.4 Degradation follows pseudo first-order kinetics and is catalyzed by various buffer salts, production medium ingredients, S. clavuligerus cells, amino acids, and different divalent metal chlorides.2,5–9 Unfortunately, literature investigations cover only a small range of CA concentrations up to 2.5 g L1 (12.6 mmol L1). However, from a process point of view, this is only a fraction of the CA concentration range that needs to be considered. Though the CA titer in fermentations of the wild type strain is only approximately 0.5 g L1 (2.5 mmol L1),8,9 in industrial fermentations employing overproducing mutants much higher concentrations of up to 10 g L1 (50 mmol L1) can be expected. Such data are
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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Figure 1. Clavulanic acid (1) and its major decomposition products: The first intermediate 1-amino-2-oxo-butan-4-ol (2) reacts further to 2,5-bis(2-hydroxyethyl)pyrazine (3), 3-carboxyethyl-2,5-bis(2-hydroxyethyl)pyrazine (4) and 3-ethyl-2,5-bis(2-hydroxyethyl)pyrazine (5). Other final products or intermediates, which are not shown, include amongst others carbon dioxide and acetaldehyde.
usually proprietary, but, for example, a final titer of 3.8 g L1 (19.1 mmol L1) was disclosed in a patent together with aqueous concentrations of 10–30 g L1 (50–150 mmol L1) during product purification.10,11 In this work we investigated the decomposition kinetics for CA concentrations of up to 20 g L1 (100 mmol L1), covering thereby a concentration range of practical importance, and can show that the degradation of CA is accelerated at higher concentrations.
MATERIALS AND METHODS All experiments were performed in aliquots of 10 mL containing different amounts of potassium clavulanate (International Laboratory, San Bruno, CA) in 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7) shaken at 288C in 15 mL closed falcon tubes. When indicated, 0.05 equivalents EDTA were added to the reaction mixture. To investigate the influence of CA decomposition products, potassium clavulanate equivalent to 20 g L1 (100 mmol L1) CA in 0.1 M MOPS buffer (pH 7) was incubated at 808C for 48 h to complete degradation. The same decomposition products could be found in this solution as in the solutions of the kinetic experiments, however JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
in a slightly different ratio (data not shown). After cooling, this solution was diluted with 0.1 M MOPS buffer and potassium clavulanate was added to final CA concentrations of 5 g L1 (25 mmol L1). Sample aliquots of 0.5 mL were removed regularly and the remaining CA concentrations were measured immediately. All experiments were performed in triplicates. The average values of each three measurements were used to calculate the decomposition constant by a curve fit with the IGOR software (Wavemetrics, Inc., Portland, OR). CA in the sample was quantified as the imidazole derivative after HPLC separation (Merck LaChrom system L-7200, VWR, Dietlikon, Switzerland) on a Prontosil Eurobond C18 5.0 mm column (Bischoff Chromatography, Leonberg, Germany) flushed with a mixture of 30% methanol and 70% 50 mM KH2PO4 (pH 4.5). The detection wavelength of the diode array detector (Merck LaChrom L-7455) was set to 312 nm.
RESULTS We investigated the influence of higher CA concentrations on its decomposition kinetics, DOI 10.1002/jps
CLAVULANIC ACID DECOMPOSITION KINETICS
3453
and concentrated on the range between 2 and 20 g L1 (10 and 100 mmol L1), which covers the probable range of titers during industrial fermentations and product purification. First, buffered solutions containing 2–20 g L1 (10–100 mmol L1) CA were incubated and the time course of CA concentration was followed (Fig. 2). As the pH remained constant at 7 throughout the experiment, the hydrolysis of CA can be described by a pseudo first order kinetics of the form d½CA ¼ kobs ½CA dt
(1)
Fitting an exponential curve to the plot of the ratio of [CA] and the initial CA concentration [CA]0 over time yields the first order decomposition constant kobs.5,6,8,9 Clearly, the decomposition Figure 3. Influence of different amount of degraded CA added at t ¼ 0 h to the reaction mixtures with [DP]0 ¼ 5 g L1 at T ¼ 288C.
Figure 2. Decomposition of CA in MOPS buffer at T ¼ 288C plotted (a) as the course of relative CA concentration with time and (b) in the logarithmic form. DOI 10.1002/jps
of CA in the more concentrated solutions is faster than in the less concentrated one (Fig. 2, Tab. 1). This implies that the varying CA]0 have a direct influence on the concentration of catalytic compounds in the solution leading to an acceleration of the reaction at higher starting concentrations. The experiment also showed that the rate constant remained approximately constant over the investigated period (Fig. 2b), suggesting that also the concentration of the catalysts remained approximately constant during the reaction. Next, we examined the possible reasons for this unexpected kinetic behavior such as acceleration by metal impurities in the CA salt. We repeated the experiments in the presence of 0.05 eq EDTA to complex divalent metal ions.12 However, the same effect as in solutions without EDTA could be observed (Tab. 1). Alternatively, CA could itself catalyze its own decomposition. However, as the catalyst would then be consumed during the reaction, but the pseudo-first order degradation constant remained essentially constant over the degradation process (Fig. 2b), we had to assume that also the degradation products exhibit a similar catalytic activity. In order to test this hypothesis, a stock solution of degraded CA, containing the same decomposition products was diluted to different concentrations between 2 and 10 g L1 (10 and 50 mmol L1, referring to the original [CA]) with MOPS buffer and CA was added to a final concentration of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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Table 1. Summary of the Observed Kinetic Constants at T ¼ 288C for the Decomposition of CA Under Different Conditions Decomposition background
[CA]0/(g L1)
kobs/h1
0.1 M MOPS buffer, pH 7
2.5 10 20 2 17.6 5 5 5
0.0038 0.0001 0.0061 0.0003 0.0086 0.0006 0.004 0.0002 0.0076 0.0003 0.0044 0.0002 0.0049 0.0001 0.0067 0.0003
0.1 M MOPS, 0.05 eq EDTA, pH 7 Decomposition products from 2 g L1 CA Decomposition products from 5 g L1 CA Decomposition products from 10 g L1 CA
5 g L1 (25 mmol L1). Indeed, we could confirm that increased amounts of degraded CA in the solution led to higher decomposition rates (Fig. 3, Tab. 1). Based on these data we propose a kinetic model which expresses kobs as a function of the catalyst concentration, as often applied for general acid– base catalysis: kobs ¼ kMOPS þ kcat ð½CA0 þ ½DP0 Þ
(2)
The catalytic constant kMOPS denotes the decomposition constant in the absence of any other catalytic species except MOPS (0.1 M, pH 7) and kcat is the catalytic constant for the sum of CA and decomposition products (DP) at t ¼ 0 h. The decomposition product concentration [DP]0 is expressed as the [CA]0 from which the degradation products were produced and is included here to be able to model those experiments for which decomposition products were added at the beginning of the reaction. In order to test this model, we inserted Eq. (2) in Eq. (1) and globally fitted the resulting equation to our complete data set to obtain values for kMOPS ((3.01 103 0.23 103) h1) and kcat ((2.58 104 0.23 104) L (g h)1, 5.16 105 4.6 106) L (mmol h)1) and compared the resulting calculated kobs with the measured decomposition constant. The measured values were in good agreement with the modeled data, supporting the validity of the model (Fig. 4).
DISCUSSION The decomposition of CA can be satisfactorily described by pseudo first order kinetics, as shown by several previous studies2,5,8,9 and also confirmed by our own measurements. We show here JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
that the observed pseudo first order decomposition constant depends on the initial CA concentration, which had not yet been accounted for in the available literature. As an effect of metal impurities could be excluded, we reasoned that CA and its degradation products catalyze the compound’s decomposition. Similar observations were made for other antibiotics. For example the hydrolysis of ceftazidime is self-accelerated at higher concentration because ceftazidime and its decomposition product act as similarly strong general-base catalysts,13 and the authors concluded that all antibiotics which are sensitive to
Figure 4. Comparison of simulated and measured kobs in buffered solutions at T ¼ 288C as a function of the sum of the initial CA concentration and the initial decomposition product concentration. The dashed line shows the simulated average kobs values and the dotted lines flank the maximal and minimal values of the simulated kobs values based on the uncertainties of kMOPS and kcat. The circles and triangles denote the measured kobs values. DOI 10.1002/jps
CLAVULANIC ACID DECOMPOSITION KINETICS
general acid–base catalysis and contain functional groups such as amino- or hydroxyl groups, which can act as such catalysts, are likely to selfcatalyze their own decomposition. Indeed, the isomerisation of ceftibuten is facilitated by itself as a general acid or base catalyst,14 so is the hydrolysis of amoxicillin by the catalytic action of the phenolic hydroxyl group15 and the degradation of imexon.16 Literature data showed that CA degradation is prone to general acid or base catalysis, as the observed decomposition constants vary with buffer and medium components whereas amino- and ammonium groups have the most pronounced effect.2,6,8,9,12 Thus it is reasonable to assume that CA and its decomposition products are able to catalyze the hydrolysis of the compound. The described model is able to predict the kinetic of the reaction in a rather reliable manner. However, the points for experiments without degradation products at the beginning lay in the upper range of the calculated area, whereas the values for the reactions supplemented with degradation product lay in the lower range. This suggests that as degradation progresses the catalytic activity of the resulting compound mixture decreases, which indicates that at least one of the chosen simplifications—constant overall catalyst concentrations and constant rate constant—would merit further investigations, indicating the limits of the chosen approach. Furthermore, this approach does neither appreciate in detail the observation that the decomposition product pattern changes with varying CA concentrations (data not shown).17 Nevertheless, the suggested model supplies a simple description of the complex degradation behavior of CA that should be considered in any process, where high aqueous CA concentrations are encountered, such as during industrial fermentations and subsequent product isolation.
REFERENCES 1. Reading C, Cole M. 1977. Clavulanic acid—blactamase-inhibiting b-lactam from Streptomyces clavuligerus. Antimicrob Agents Chemother 11: 852–857. 2. Haginaka J, Nakagawa T, Uno T. 1981. Stability of clavulanic acid in aqueous solutions. Chem Pharm Bull 29:3334–3341.
DOI 10.1002/jps
3455
3. Haginaka J, Yasuda H, Uno T, Nakagawa T. 1985. Degradation of clavulanic acid in aqueous alkaline solution—Isolation and structural investigation of degradation products. Chem Pharm Bull 33:218– 224. 4. Finn MJ, Harris MA, Hunt E, Zomaya II. 1984. Studies on the hydrolysis of clavulanic acid. J Chem Soc Perkin Trans 1:1345–1349. 5. Bersanetti PA, Almeida R, Barboza M, Araujo MLG, Hokka CO. 2005. Kinetic studies on clavulanic acid degradation. Biochem Eng J 23:31–36. 6. Ishida K, Hung TV, Lee HC, Liou K, Shin CH, Yoon VJ, Sohng JK. 2006. Degradation of clavulanic acid during the cultivation of Streptomyces clavuligerus; instability of clavulanic acid by metabolites and proteins from the strain. J Microbiol Biotechnol 16:590–596. 7. Martin J, Mendez R, Salto F. 1989. Studies on clavulanic acid 2: The catalytic effect of metal-ions on the hydrolysis of clavulanic acid. J Chem Soc Perkin Trans 2:227–231. 8. Mayer AF, Deckwer WD. 1996. Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations. Appl Microbiol Biotechnol 45:41–46. 9. Roubos JA, Krabben P, de Laat W, Babuska R, Heijnen JJ. 2002. Clavulanic acid degradation in Streptomyces clavuligerus fed-batch cultivations. Biotechnol Prog 18:451–457. 10. Cardoso JP. 2002. Process for the isolation of a phamaceutically acceptable alkali metal salt of clavulanic acid. US 6,417, 352. 11. Fleming ID, Noble D, Noble HM, Wall WF. 1984. Pure salts of clavulanic acid, e.g. lithium clavulanate. US 4,490, 294. 12. Martin J, Mendez R, Alemany T. 1989. Studies on clavulanic acid 1: Stability of clavulanic acid in aqueous solutions of amines containing hydroxy groups. J Chem Soc Perkin Trans 2:223–226. 13. Fubara JO, Notari RE. 1998. A kinetic oxymoron: Concentration-dependent first order rate constants for hydrolysis of ceftazidime. J Pharm Sci 87:53–58. 14. Hashimoto N, Hirano K. 1998. Isomerization of ceftibuten in aqueous solution. J Pharm Sci 87:1091–1096. 15. Bundgaard H. 1977. Polymerization of penicillins 2. Kinetics and mechanism of dimerization and selfcatalyzed hydrolysis of amoxycillin in aqueous solution. Acta Pharm Suec 14:47–66. 16. Kuehl PJ, Hoye WL, Myrdal PB. 2006. Preformulation studies on Imexon. Drug Dev Ind Pharm 32:687–697. 17. Brethauer S. 2007. In situ product removal as an approach to improve the fermentative production of clavulanic acid. PhD Thesis, ETH Zurich.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
Received 21 June 2007; revised 31 August 2007; accepted 14 September 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21225
ABSTRACT: The decomposition kinetics of the b-lactamase inhibitor clavulanic acid (CA) was investigated for CA concentrations between 2.5 and 20 g L1, which is assumed to represent a characteristic range for an industrial CA production process. For each initial concentration, first order kinetics plots could be obtained, however the kinetic constant increased from 3.8 103 to 8.6 103 h1 with increasing initial CA concentration, indicating that CA accelerates its own decomposition by general acid–base catalysis. Furthermore, the kinetic constant remained approximately constant during the reaction, suggesting that also the decomposition products of CA had to show similar catalytic activity. This was confirmed experimentally by increased CA decomposition rates when CA degradation products were added to the reaction. A kinetic model is proposed, which is able to reliably predict the observed pseudo first order rate constants. The presented results should be considered in any process where highly concentrated CA solutions are employed, for example, during final downstream processing or in industrial fermentations. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3451–3455, 2008
Keywords:
chemical stability; kinetics; anti-infectives; HPLC; mathematical model
INTRODUCTION Clavulanic acid (CA) (1) ((2R,3Z,5R)-3-(2-hydroxyethyliden)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptan-2carboxylic acid) is a fused bicylic b-lactam molecule produced by Streptomyces clavuligerus. CA is one of the active ingredients in Augmentin1 and acts as a potent inhibitor of b-lactamases produced by some b-lactam resistant pathogenic microorganism to enable efficient treatment of infectious diseases.1 The production as well as the downstream processing is accompanied by the compound’s degradation, initiated by hydrolysis, which is around 10 times faster than the one of penicillin G.2 Correspondence to: Sven Panke (Telephone: þ41-44-632-0413; Fax: þ41-44-632-19-93; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 3451–3455 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
Hydrolysis of CA under neutral or basic conditions proceeds via the reactive amino ketone (2) to pyrazine end-products (3–5) (Fig. 1),3,4 which are part of a complex product mixture.4 Degradation follows pseudo first-order kinetics and is catalyzed by various buffer salts, production medium ingredients, S. clavuligerus cells, amino acids, and different divalent metal chlorides.2,5–9 Unfortunately, literature investigations cover only a small range of CA concentrations up to 2.5 g L1 (12.6 mmol L1). However, from a process point of view, this is only a fraction of the CA concentration range that needs to be considered. Though the CA titer in fermentations of the wild type strain is only approximately 0.5 g L1 (2.5 mmol L1),8,9 in industrial fermentations employing overproducing mutants much higher concentrations of up to 10 g L1 (50 mmol L1) can be expected. Such data are
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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Figure 1. Clavulanic acid (1) and its major decomposition products: The first intermediate 1-amino-2-oxo-butan-4-ol (2) reacts further to 2,5-bis(2-hydroxyethyl)pyrazine (3), 3-carboxyethyl-2,5-bis(2-hydroxyethyl)pyrazine (4) and 3-ethyl-2,5-bis(2-hydroxyethyl)pyrazine (5). Other final products or intermediates, which are not shown, include amongst others carbon dioxide and acetaldehyde.
usually proprietary, but, for example, a final titer of 3.8 g L1 (19.1 mmol L1) was disclosed in a patent together with aqueous concentrations of 10–30 g L1 (50–150 mmol L1) during product purification.10,11 In this work we investigated the decomposition kinetics for CA concentrations of up to 20 g L1 (100 mmol L1), covering thereby a concentration range of practical importance, and can show that the degradation of CA is accelerated at higher concentrations.
MATERIALS AND METHODS All experiments were performed in aliquots of 10 mL containing different amounts of potassium clavulanate (International Laboratory, San Bruno, CA) in 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7) shaken at 288C in 15 mL closed falcon tubes. When indicated, 0.05 equivalents EDTA were added to the reaction mixture. To investigate the influence of CA decomposition products, potassium clavulanate equivalent to 20 g L1 (100 mmol L1) CA in 0.1 M MOPS buffer (pH 7) was incubated at 808C for 48 h to complete degradation. The same decomposition products could be found in this solution as in the solutions of the kinetic experiments, however JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
in a slightly different ratio (data not shown). After cooling, this solution was diluted with 0.1 M MOPS buffer and potassium clavulanate was added to final CA concentrations of 5 g L1 (25 mmol L1). Sample aliquots of 0.5 mL were removed regularly and the remaining CA concentrations were measured immediately. All experiments were performed in triplicates. The average values of each three measurements were used to calculate the decomposition constant by a curve fit with the IGOR software (Wavemetrics, Inc., Portland, OR). CA in the sample was quantified as the imidazole derivative after HPLC separation (Merck LaChrom system L-7200, VWR, Dietlikon, Switzerland) on a Prontosil Eurobond C18 5.0 mm column (Bischoff Chromatography, Leonberg, Germany) flushed with a mixture of 30% methanol and 70% 50 mM KH2PO4 (pH 4.5). The detection wavelength of the diode array detector (Merck LaChrom L-7455) was set to 312 nm.
RESULTS We investigated the influence of higher CA concentrations on its decomposition kinetics, DOI 10.1002/jps
CLAVULANIC ACID DECOMPOSITION KINETICS
3453
and concentrated on the range between 2 and 20 g L1 (10 and 100 mmol L1), which covers the probable range of titers during industrial fermentations and product purification. First, buffered solutions containing 2–20 g L1 (10–100 mmol L1) CA were incubated and the time course of CA concentration was followed (Fig. 2). As the pH remained constant at 7 throughout the experiment, the hydrolysis of CA can be described by a pseudo first order kinetics of the form d½CA ¼ kobs ½CA dt
(1)
Fitting an exponential curve to the plot of the ratio of [CA] and the initial CA concentration [CA]0 over time yields the first order decomposition constant kobs.5,6,8,9 Clearly, the decomposition Figure 3. Influence of different amount of degraded CA added at t ¼ 0 h to the reaction mixtures with [DP]0 ¼ 5 g L1 at T ¼ 288C.
Figure 2. Decomposition of CA in MOPS buffer at T ¼ 288C plotted (a) as the course of relative CA concentration with time and (b) in the logarithmic form. DOI 10.1002/jps
of CA in the more concentrated solutions is faster than in the less concentrated one (Fig. 2, Tab. 1). This implies that the varying CA]0 have a direct influence on the concentration of catalytic compounds in the solution leading to an acceleration of the reaction at higher starting concentrations. The experiment also showed that the rate constant remained approximately constant over the investigated period (Fig. 2b), suggesting that also the concentration of the catalysts remained approximately constant during the reaction. Next, we examined the possible reasons for this unexpected kinetic behavior such as acceleration by metal impurities in the CA salt. We repeated the experiments in the presence of 0.05 eq EDTA to complex divalent metal ions.12 However, the same effect as in solutions without EDTA could be observed (Tab. 1). Alternatively, CA could itself catalyze its own decomposition. However, as the catalyst would then be consumed during the reaction, but the pseudo-first order degradation constant remained essentially constant over the degradation process (Fig. 2b), we had to assume that also the degradation products exhibit a similar catalytic activity. In order to test this hypothesis, a stock solution of degraded CA, containing the same decomposition products was diluted to different concentrations between 2 and 10 g L1 (10 and 50 mmol L1, referring to the original [CA]) with MOPS buffer and CA was added to a final concentration of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
3454
BRETHAUER, HELD, AND PANKE
Table 1. Summary of the Observed Kinetic Constants at T ¼ 288C for the Decomposition of CA Under Different Conditions Decomposition background
[CA]0/(g L1)
kobs/h1
0.1 M MOPS buffer, pH 7
2.5 10 20 2 17.6 5 5 5
0.0038 0.0001 0.0061 0.0003 0.0086 0.0006 0.004 0.0002 0.0076 0.0003 0.0044 0.0002 0.0049 0.0001 0.0067 0.0003
0.1 M MOPS, 0.05 eq EDTA, pH 7 Decomposition products from 2 g L1 CA Decomposition products from 5 g L1 CA Decomposition products from 10 g L1 CA
5 g L1 (25 mmol L1). Indeed, we could confirm that increased amounts of degraded CA in the solution led to higher decomposition rates (Fig. 3, Tab. 1). Based on these data we propose a kinetic model which expresses kobs as a function of the catalyst concentration, as often applied for general acid– base catalysis: kobs ¼ kMOPS þ kcat ð½CA0 þ ½DP0 Þ
(2)
The catalytic constant kMOPS denotes the decomposition constant in the absence of any other catalytic species except MOPS (0.1 M, pH 7) and kcat is the catalytic constant for the sum of CA and decomposition products (DP) at t ¼ 0 h. The decomposition product concentration [DP]0 is expressed as the [CA]0 from which the degradation products were produced and is included here to be able to model those experiments for which decomposition products were added at the beginning of the reaction. In order to test this model, we inserted Eq. (2) in Eq. (1) and globally fitted the resulting equation to our complete data set to obtain values for kMOPS ((3.01 103 0.23 103) h1) and kcat ((2.58 104 0.23 104) L (g h)1, 5.16 105 4.6 106) L (mmol h)1) and compared the resulting calculated kobs with the measured decomposition constant. The measured values were in good agreement with the modeled data, supporting the validity of the model (Fig. 4).
DISCUSSION The decomposition of CA can be satisfactorily described by pseudo first order kinetics, as shown by several previous studies2,5,8,9 and also confirmed by our own measurements. We show here JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
that the observed pseudo first order decomposition constant depends on the initial CA concentration, which had not yet been accounted for in the available literature. As an effect of metal impurities could be excluded, we reasoned that CA and its degradation products catalyze the compound’s decomposition. Similar observations were made for other antibiotics. For example the hydrolysis of ceftazidime is self-accelerated at higher concentration because ceftazidime and its decomposition product act as similarly strong general-base catalysts,13 and the authors concluded that all antibiotics which are sensitive to
Figure 4. Comparison of simulated and measured kobs in buffered solutions at T ¼ 288C as a function of the sum of the initial CA concentration and the initial decomposition product concentration. The dashed line shows the simulated average kobs values and the dotted lines flank the maximal and minimal values of the simulated kobs values based on the uncertainties of kMOPS and kcat. The circles and triangles denote the measured kobs values. DOI 10.1002/jps
CLAVULANIC ACID DECOMPOSITION KINETICS
general acid–base catalysis and contain functional groups such as amino- or hydroxyl groups, which can act as such catalysts, are likely to selfcatalyze their own decomposition. Indeed, the isomerisation of ceftibuten is facilitated by itself as a general acid or base catalyst,14 so is the hydrolysis of amoxicillin by the catalytic action of the phenolic hydroxyl group15 and the degradation of imexon.16 Literature data showed that CA degradation is prone to general acid or base catalysis, as the observed decomposition constants vary with buffer and medium components whereas amino- and ammonium groups have the most pronounced effect.2,6,8,9,12 Thus it is reasonable to assume that CA and its decomposition products are able to catalyze the hydrolysis of the compound. The described model is able to predict the kinetic of the reaction in a rather reliable manner. However, the points for experiments without degradation products at the beginning lay in the upper range of the calculated area, whereas the values for the reactions supplemented with degradation product lay in the lower range. This suggests that as degradation progresses the catalytic activity of the resulting compound mixture decreases, which indicates that at least one of the chosen simplifications—constant overall catalyst concentrations and constant rate constant—would merit further investigations, indicating the limits of the chosen approach. Furthermore, this approach does neither appreciate in detail the observation that the decomposition product pattern changes with varying CA concentrations (data not shown).17 Nevertheless, the suggested model supplies a simple description of the complex degradation behavior of CA that should be considered in any process, where high aqueous CA concentrations are encountered, such as during industrial fermentations and subsequent product isolation.
REFERENCES 1. Reading C, Cole M. 1977. Clavulanic acid—blactamase-inhibiting b-lactam from Streptomyces clavuligerus. Antimicrob Agents Chemother 11: 852–857. 2. Haginaka J, Nakagawa T, Uno T. 1981. Stability of clavulanic acid in aqueous solutions. Chem Pharm Bull 29:3334–3341.
DOI 10.1002/jps
3455
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