Abstract

Amoxycillin is a -lactam antibiotic that is frequently used in human and

veterinary medicine due to its low cost and broad spectrum activity.
Unfortunately, bacterial resistance to this antibiotic has been increasing in
prevalence, with many strains now resistant to amoxycillin. The most
common form of resistance to -lactam antibiotics is via production of lactamases. These -lactamases act to destroy the -lactam ring of
amoxycillin, rendering it inert. In order to combat this growing problem,
amoxycillin is often combined with clavulanic acid, a -lactamase
inhibitor. This agent inhibits the action of -lactamases, protecting the
amoxycillin and allowing it to destroy the bacteria. The following article
reviews

the

chemistry,

pharmacodynamics,

metabolism of this highly effective combination.

pharmacokinetics

and

Introduction
Amoxycillin is a -lactam antibiotic that is frequently used in human and veterinary
medicine due to its low cost and broad spectrum activity. Its bacteriocidal action inhibits
the biosynthesis of bacterial cell walls by binding to the enzymes responsible for
producing the cell wall (Reyns et al, 2008).

The -lactam ring is required for

antibacterial action; however it is susceptible to inactivation by -lactamases. lactamase is produced by bacteria and acts to destroy the -lactam ring of amoxycillin.
The problem of bacterial resistance to -lactams is increasing in prevalence due to
widespread exposure over the last 50 years and the continuous evolution of -lactamases
(Miller et al, 2001).
The use of -lactamase inhibitors in combination with -lactams has been a highly
successful approach to combating this form of resistance (Miller et al, 2001). One
example of this type of inhibitor is clavulanic acid which was isolated from Streptomyces
clavuligerus in the 1970s. It is a powerful irreversible inhibitor of most -lactamases
which allows the dose of amoxycillin to be decreased while increasing its spectrum of
activity (Patrick, 2005).
To this date, development and use of -lactam/ -lactamase inhibitor combinations has
been the subject of much research. In the future more research is required to develop
new -lactamase inhibitors capable of combating the continually changing epidemiology
of -lactamases (Miller et al, 2001).
The chemistry, pharmacokinetics, pharmacodynamics and metabolism of amoxycillin and
clavulanic acid will be discussed in detail in this report.
Chemistry of Amoxycillin and Clavulanic Acid
Amoxycillin
The basic structure of penicillins consists of a thiazolidine ring (A) connected to a lactam ring (B) to which is attached a side chain (R). The penicillin nucleus itself is the
chief structural requirement for biological activity; metabolic transformation or chemical

alteration of this portion of the molecule causes loss of all significant antibacterial
activity (see figure 1.)(Goodman & Gillman, 2005).

Figure 1. Basic structure of penicillins with the -lactam ring essential for activity

Amino-penicillins are semisynthetic derivatives of penicillin that are produced by

acylation of 6-aminopenicillanic acid (6-APA). They contain a polar group which gives
rise to enhanced activity against gram-negative bacteria compared with natural penicillins
and penicillinase-resistant penicillins (AHFS Drug Information [online]).
Amoxycillin is an amino-penicillin that is structurally similar to ampicillin but has a
hydroxyl group on the phenyl ring at R1 (see figure 2.)

Figure 2. Structure of Amoxycillin (left) and differences between ampicillin and

amoxycillin (right) Ampicillin: R = H Amoxycillin: R = OH. (taken
from Goodman & Gillman, 2005)

Amoxycillin appears as a white, practically odourless, crystalline powder.

It has a

molecular weight of 419.45, pKa of 2.8,7.4 and 9.6 and is slightly soluble in water (1 in
400) and in methanol (1 in 200). It is insoluble in carbon tetrachloride and in chloroform.
Amoxycillin is lipophilic so that it can cross through membranes of gram-positive
bacteria.
Amoxycillin has an electron-withdrawing group with a polar hydroxyl group added to the
6-position amide (located on the -lactam ring). This increases its spectrum of action
(active against both gram negative and gram positive bacteria) as the polar groups allow
the drug to cross the gram-negative cell wall through porins. The addition of this
functional group also increases acid stability (by making the amide oxygen less
nucleophilic) when comparing it to other penicillins, like ampicillin. This does not
protect the -lactam ring from -lactamases, which are produced by resistant bacteria
and render the antibiotic inactive (Beleh, M., 2006).
Mechanism of Action
Amoxycillin's mechanism of action involves the inhibition of stage III of the bacterial
cell wall synthesis, more specifically preventing cross-linking of peptidoglycan. It
provides an alternative substrate for transpeptidases; this is because of its structural
similarity to the transition state of the Ala-Ala terminal during cross-linking. The
transpeptidases are involved with the cross-linking of the peptidoglycan layer that is a
major and minor component of gram positive and gram-negative cell wall respectively.
The -lactam ring in amoxycillin is unstable and reacts with a serine residue of the
transpeptidase (shown in figure 3). This reaction is irreversible which prevents the further
growth of the bacterial cell wall. The resulting complex is stable to water and remains
attached to the polypeptide chain (Silverman, 1992). This leads to lysis of the cell, and as
such amoxycillin has an bactericidal effect on susceptible bacteria. The secondary amine
on the C6 can be protonated and the phenol group can be deprotonated. The carboxylic
acid at position 3 is also capable of deprotonation. When these groups are ionised, the
molecule contains a net negative charge and therefore is less Zwitterionic and more
capable of being orally absorbed.

Figure 3. How penicillins bind to bacterial proteins (taken from Penicillin Mechanism [online])

Stability
Amoxycillin is more resistant to acid-catalysed hydrolysis than natural penicillins and is
generally stable in the presence of acidic gastric secretions following oral administration.
Amoxycillin has a half-live of 1520 hours in solution with a pH of 2 at 35C (AHFS
Drug Information [online]).

Figure 4. Mechanism of -lactamase on the -lactam ring of penicillins (taken from Beta-Lactamases
[online]).

Resistance to amoxycillin is due to the production of -lactamase enzymes by resistant

bacteria. -lactamases attack the -lactam ring at the carbonyl position (see figure 4.)
opening the -lactam and thus inactivating amoxycillin (Foyes Med. Chem, 2002). To
avoid this amoxycillin is prepared in combination products that include the -lactamase
inhibitor, clavulanic acid. This inhibition is irreversible and is known as suicide
inhibition. This allows amoxycillin to produce antibacterial effects.
Clavulanic Acid
Clavulanic acid is produced by Streptomyces clavuligerus;

Figure 5. Structure of clavulanic acid

Clavulanate potassium appears as a white to off-white powder and is moisture-sensitive.

It has a molecular weight of 237.25, pKa of 2.7 and is freely soluble in water, but stability
in aqueous solution is not good; optimum stability at a pH of 6.0 to 6.3; soluble in
methanol, with decomposition. As an antibiotic it has poor antimicrobial activity, but it is
a "suicide" inhibitor that irreversibly binds -lactamases.
Mechanism of Action
Clavulanic acid is believed to acylate the active site serine by mimicking the normal
substrate (Foyes Med. Chem, 2002). While hydrolysis can still occur with some lactamases, generally they are inhibited irreversibly. This leads to its classification as a
mechanism-based inhibitor or so called suicide substrate (see figure 6). When clavulanic
acid is added to amoxycillin preparations the potency against -lactamase producing
strains is markedly enhanced.

Figure 6. Mechanism of action of Clavulanic acid (taken from -lactamase inhibitors [online]).

Pharmacodynamics of Amoxycillin and Clavulanic Acid

Amoxycillin
Microbiological killing by Amoxycillin (and indeed all B-lactams) is a time dependant
process. For effective microbial eradication of susceptible organisms, free serum
concentrations of Amoxycillin must exceed the MIC (minimum inhibitor concentration)
for at least 40% of the dosing interval. (Smith, 2003). Additionally, the PAE (postantibacterial effect) of amoxycillin is minimal (1.5-2 hours for straphylococci and
streptococci) for gram positive organisms and zero for gram negative organisms.
Increases in peak concentration of amoxycillin do not increase the PAE significantly.
(Navarro 2005) These pharmacodynamic parameters have profound implications on the
way Amoxycillin is dosed in clinical practice. Dosing strategies for Amoxycillin are
designed to maintain serum concentrations of Amoxycillin above the MIC of susceptible
organisms for the maximum possible time. As a result, dosing intervals are shortened to
twice or three times a day.
It is important to understand the resistance mechanisms of targeted microorganisms to
amoxycillin, in order to relate the pharmacodynamics of these mechanisms to dosing of
amoxycillin. Microbial resistance to amoxycillin is expedited by the production of lactamases. These -lactamases are produced by the targeted organism and act to cleave
the -lactam ring of amoxycillin. Specifically, an active site serine on the -lactamase
performs a nucleophillic attack on the lactam ring of amoxycillin, opening it and
rendering it ineffective. (Wright, 2005) To minimise the extent of microbial resistance to
amoxycillin, dosage regimes are kept short, and use high doses of active drug.
Cmax/MIC ratios of 3-4 (Navarro. 2005) are most appropriate and correspond to high
doses of amoxycillin, taken regularly throughout the day. This reduces the risk of
resistance forming, but does not eradicate it entirely.
The pharmacodynamics of amoxycillin, in addition to the development of resistance,
necessitates a dosing regimen that is both high dose and short interval. Additionally,
increasing resistance remains a problem, with many amoxycillin resistant strains now
prevalent. The addition of clavulanic acid to amoxycillin preparations reduces the
problem of microbial resistance and allows for more effective microbial eradication and a
wider spectrum of action.

Clavulanic Acid
Clavulanic acid is a -Lactamase inhibitor which possesses little intrinsic antibacterial
activity. It inhibits the action of -lactamase by forming a suicide substrate, which
irreversibly deactivates the enzyme. Specifically, clavulanic acid interacts with Ser-70 of
the enzyme, forming an unstable intermediate, which is converted to an inactive, stable
aldehyde. This deactivation of -lactamase protects the amoxycillin from degradation,
allowing it to eliminate susceptible bacteria. (Sandanayaka, Prashad. 2002)
The result of the addition of clavulanic acid to amoxycillin preparations is a lowered MIC
in microorganisms which confer their resistance via -lactamases. For example the MIC
of amoxycillin against Haemophilus influenzae is >64mg/L. This bacterium produces lactamase, which increases the MIC significantly. With the addition of clavulanic acid,
the MIC falls to 1.0mg/L. (Lee, et al. 2003) As such, lower doses of amoxycillin can be
used to maintain a T>MIC of 40% or more.
Unfortunately, the widespread use of Amoxycillin/Clavulanic acid combinations has been
against the backdrop of increasing antibiotic resistance. With the MICs of many strains
becoming too high for effective treatment with current combinations, new,
pharmacodynamically and pharmacokinetically enhanced formulations have been
developed.

For

example,

new

paediatric

formulation

uses

dose

of

(amoxycillin/clavulanic acid) 90mg/6.4mg per kg/day. This represents a doubling in the

dose of amoxycillin compared to previous formulations and the same dose of clavulanic
acid. With this new formulation, bacteria with increased MICs can be effectively treated,
as the T>MIC remains above 40% for these bacteria. (Navarro, 2005)
Pharmacokinetics of Amoxycillin and Clavulanic Acid
Pharmacokinetics
Clavulanic acid appears to be active against a wide spectrum of gram-positive and
negative bacteria, but the activity is low compared to other antibiotics so it often
inappropriate to use in isolation. (Parag et al., 2008)
The pharmacokinetic (PK) rationale for combining amoxycillin with fixed dose
clavulanic acids (125mg) is based on their similar elimination half-lives(~1h) and
volumes of distribution (Vclavulanic

acid

/Vamoxycillin= 0.85 after IV infusion). Additionally, an

excess dose of clavulanic acid is used to inhibit -lactamase produced by resistant

bacterial strains. (Vree et al., 2003, Parag et al., 2008)
Vree et al. selected 144 healthy Caucasian male subjects with normal renal function to
investigate the pharmacokinetics of amoxycillin with clavulanic acid at different doses
and formulations. Data were obtained from plasma concentration-time (over 12h after
dosing) curves from four different open, randomized, two-treatment (separated by a 1week washout period) crossover Phase I bioequivalence studies, with following CoamoxiclavTM (A) and AugmentinTM (B) formulations: tablets 250/125 (A1/B1), 500/125
(A2/B2) and 875/125 (A4/B4) mg, or 10mL (A3/B3) of an oral suspension
(250/62.5mg/5mL). (Vree et al., 2003)

Figure 7. Mean(S.D.) plasma concentrationtime curves of amoxycillin (Amoxi) and clavulanic acid
(Clav) after an oral dose of 500/125mg (A2) co-amoxiclav tablets in 36 healthy volunteers. (Vree et al.,
2003)

The elimination t1/2 of amoxycillin (~1.46 h), and that of clavulanic acid (~1.08 h) was
not only for 500/125 co-amoxiclav tablets but for all other dosages and formulations (A1A4 and B1-B4) tested. (Vree et al., 2003)
Studies have also shown that the absorption of each formulation was fast, although the
Tmax (time to reach maximum concentration) of amoxycillin increased with the dose,
from 1.14 0.41 h at 250 mg to 2.041.01 h (P < 0.0001) at 875 mg, which would
suggest a rate limiting step in the amoxycillin absorption process. (Vree et al., 2003, 3, 4)
No differences in the pharmacokinetic parameters of clavulanic acid (fixed dose 125mg)

were seen among the different formulations. However, variations (figure-2) in the AUC t
ratio of amoxycillin/clavulanic acid (2:1-10:1) were observed and highlight the variable
nature of clavulanic acid absorption when combined with amoxycillin. In other studies
where clavulanic acid was administered alone, the mean absorption was 97% with little
inter-patient variability, suggesting an interaction between the absorption of amoxycillin
and clavulanic acid. (Allen et al., 1998) But all clinical data suggested that such
variability does not affect the efficacy of the combined products and that the current
dosage ratio of 4:1 may be considered as conservative. (Vree et al., 2003, Natsch et al).
1998)

Figure 8. Mean AUCts of amoxycillin plotted versus the mean AUCts of clavulanic acid for the oral
administrations (formulation A) of co-amoxiclav suspension (), and oral tablets 875/125 mg (), 500/125
mg (), and 250/125 mg (). Dotted lines represent 95% confidence intervals. Similar results were
obtained for formulation B. (Vree et al., 2003)

In figure-8, the AUCamox/AUCclav regression curve of amoxycillin 875 mg has a negative

slope, which may indicate that the saturable absorption for amoxycillin is influenced by
the presence of clavulanic acid. In contrast, the slopes of the regression lines of the
250/125 and 500/125 mg dosages were all positive, indicating no influence of clavulanic
acid administration on the absorption of amoxycillin at these dosages. (Vree et al., 2003)
Not only important in determining the absorption and elimination profiles,
pharmacokinetics of amoxycillin/clavulanic acid was also used as a unique approach to
develop PK enhanced formulations, which were aimed at eradicating S. pneumoniae,

including penicillin-resistant strains with amoxycillin minimum inhibitory concentrations

(MICs) as high as 4 mg/L. (Kaye et al., 2001)
Human PK and variability were determined along with the MIC distribution of the target
pathogens, and modified dosage regimens were simulated to produce sufficient target
attainments. PK and pharmacodynamic principles provide a mechanism to correlate in
vitro potency with in vivo efficacy of antibiotics. For -lactams, such as amoxycillin, the
unbound serum concentration of the drug exceeding the MIC of the causative pathogen
for 4050% of the dosing interval is predictive of efficacy (bacterial eradication) and can
be used to determine the PK breakpoint (4mg/L) for that specific dosing regimen.
(Jacobs., 2007)
This approach has led to the development of a high-dose pediatric formulation of
amoxycillin/clavulanate

(Augmentin

ES-600)

in

which

the

daily

dose

of

amoxycillin/clavulanate was increased from 45/6.4 mg/kg/day to 90/6.4 mg/kg/day,

extending the concentration present for 40% of the dosing interval from 2 mg/L to 4
mg/L. For adults a sustained release formulation of amoxycillin/clavulanate (Augmentin
SR), containing 2000 mg immediate and sustained release amoxycillin in one tablet,
extended the serum concentration of amoxycillin above the target MIC of 4 mg/L for
>40% of the dosing interval as shown in figure-9.(Jacobs., 2007, Kaye et al., 2001)

Figure 9. Amoxycillin concentration-time profiles after oral administration of amoxycillin/clavulanate

formulations containing 2000 mg immediate release amoxycillin and 2000 mg a pharmacokinetically
enhanced formulation containing 1125 mg of immediate and 875 mg of sustained release amoxycillin.
(Kaye et al., 2001)

Metabolism and Excretion

Amoxycillin
The amide bond of the -lactam ring is most susceptible to degradation in penicillins as it
is highly strained and reactive. This bond cleaves rapidly in alkaline solutions to produce
penicilloic acid (amoxicilloic acid in the case of amoxycillin). This reaction is essentially
irreversible and deactivates the antibiotic activity of the molecule (Foye, 2002).
Hydrolysis occurs in the liver as well as being catalysed by bacterial -lactamase (see
Figure 4.). Alcohols and amines produce the same cleavage reaction resulting in esters
and amides respectively. When proteins provide the nucleophiles involved in these
reactions, antigenic conjugates responsible for allergy to penicillins are produced (Foye,
2002).
In acidic solutions, hydrolysis of the -lactam ring is more complex due to involvement
of the -R sidechain. The main products of acid hydrolysis are penicillamine, penicilloic
acid and penilloaldehyde(Foye, 2002). The two major metabolites of amoxycillin are
amoxicilloic acid and amoxycillin diketopiperazine-2,5-dione (Reyns et al 1, 2008).
Around 70% of amoxycillin is excreted unchanged in urine. Concurrent administration
of probenecid can delay the excretion of amoxycillin and increase its plasma
concentration 2-4 fold, thus extending its therapeutic effect (MIMS, 2008). Probenecid
blocks facilitated transport of amoxycillin through the kidney tubules as well as
competing for binding sites on albumin. This occurs due to probenecid having similar
properties to amoxycillin i.e. they are both moderately lipophilic carboxylic acids
(Patrick, 2005).
Clavulanic acid
Clavulanic acid undergoes hepatic metabolism (Merck, 2008). The major metabolites are
2,5-dihydro-4-(2- hydroxyethyl)-5- oxo-1H-pyrrole-3- carboxylic acid and 1-amino-4hydroxy-butan-2-one. Small amounts of metabolites that have yet to be identified are
also shown to be present (MIMS, 2008). 30-40% of clavulanic acid is excreted in urine
unchanged in the first 6 hours after administration (MIMS, 2008).

Conclusion
Amoxycillin with clavulanic acid has been studied extensively and has show great
success against a wide spectrum of -lactamase producing bacteria after nearly 20 years
of clinical use. Different classes of -lactamases are broadly grouped into class A, B, C
and D based on their amino acid sequences and their number is increasing. There are
more clinically isolated strains showing resistance to current combined -lactam/lactamase inhibitor products. This is due to the fact that the prevalence and continuing
evolution of bacteria producing -lactamases is also increasing. While several
compounds have shown promise in inhibition of both class A and C -lactamases, none of
them have yet made it to clinical trials. Hence, second-generation -lactamase inhibitors,
which inhibit both class A and C -lactamases, and are combined with an appropriate lactam partner, are still awaiting research and testing to bring them into clinical use in the
future. (Miller., 2001) Until such time, amoxycillin combined with clavulanic acid will
remain a mainstay of therapeutics in modern medicine.

References
AHFS Drug Information (2008) Amoxycillin and Clavulanate Potassium [online]
Viewed 16/08/08
Available:
http://www.medicinescomplete.com.ezproxy1.library.usyd.edu.au/mc/ahfs/current/a385024.htm?q=
%22amoxycillin%22#_hit
Allen, G. D., Coates, P. E. & Davies, B. E., On the absorption of clavulanic acid, Biopharmaceutics and
Drug Disposition, (1998), 9, 127-136.
Augmentin Prescribing Information, MIMSOnline 2008, last updated 13/09/06, CMPMedica, St Leonards
Beleh, M. (2006) Principles of Medicinal Chemistry. Class notes.
Beta-Lactamase inhibitors [online]
viewed: 16/08/08
Available: http://homepage.ntlworld.com/diamonddove/08_BLInhibitors/BLInhibitors.htm
Beta-Lactamases [online]
viewed: 16/08/08
Available:
http://homepage.ntlworld.com/diamonddove/07_BLactamases/BLactamases.htm
Brunton, L.L, Lazo, J.S, Parker, K.L: (2005) Goodman & Gilmans The Pharmacological Basis of
Therapeutics, 11th Ed. Chapter 44

Jacobs, M., Combating resistance: application of the emerging science of pharmacokinetics and
pharmacodynamics, International Journal of Antimicrobial Agents, (2007), 30S, S122S126
Kaye CM, Allen A, Perry S, the clinical pharmacokinetics of a new pharmacokinetically enhanced
formulation of amoxycillin/clavulanate, Clinical Therapy, (2001), 23, 578584.
Lee N, Yuen K, Kumana C. Clinical Role of -Lactam/-Lactamase Inhibitor combinations. Drugs
2003;63(14):1511-1524
Miller, L., Ratnam, K. and Payne, D., b-Lactamase-inhibitor combinations in the 21st century: current
agents and new developments, Current Opinion in Pharmacology, (2001), 1: 451- 458
Natsch, S. & van der Meer, J. W., Dosing of amoxycillin/clavulanate for treatment of lower respiratory tract
infection, Clinical Infectious Diseases, (1998), 26, 529530.
Navarro A. New formulations of Amoxycillin/Clavulanic Acid. Clin Pharmacokinetics 2005;44(11):10971115
Nilsson-Ehle, I., Fellner, H., Hedstrm, S. A., Nilsson-Ehle, P. & Sjvall, J., Pharmacokinetics of
clavulanic acid, given in combination with amoxycillin, in volunteers, (1985), Journal of Antimicrobial
Chemotherapy 16, 491498
Parag S. Saudagar, Shrikant A. Survase, Rekha S. Singhal, Clavulanic acid: A review, Biotechnology
Advances, (2008), 26, 335351
Patrick, G., 2005, An Introduction to Medicinal Chemistry, 3rd edn, Oxford University Press, Oxford
Reyns, T., Cherlet, M., De Baere, S., De Backer, P. and Croubels, S. 2008, Rapid method for the
quantification of amoxycillin and its major metabolites in pig tissues by liquid chromatography-tandem
mass spectrometry with emphasis on stability issues, Journal of Chromatography B: Analytical
Technologies in the Biomedical & Life Sciences, vol. 861, no.1, pp. 108-16.
Reyns, T., Cherlet, M., De Boever, S., De Baere, S., De Backer, P. and Croubels, S. 2008, Tissue depletion
of amoxycillin and its major metabolites in pigs: influence of the administration route and the simultaneous
dosage of clavulanic acid, Journal of Agricultural & Food Chemistry, vol. 56, no. 2, p. 448-54
Silverman, R.B (1992). The Organic Chemistry of Drug Design and Drug Action, IL, USA: Academic
Press, 1st Ed. p181.
Sandanayaka V, Prashad A. Resistance to -Lactam antibiotics: Structure and Mechanism based design of
-Lactamase Inhibitors. Current Medicinal Chemistry 2002; 9:1145-1165
Smith C. Pharmacokinetic/Pharmacodynamic Considerations in Community-Aquired Lower Respiratory
Tract Infections. Journal of Pharmacy Practice. 2003;16(5):313-323
Vree, T., Dammers, E. and Exler, P., Identical pattern of highly variable absorption of clavulanic acid from
four different oral formulations of co-amoxiclav in healthy subjects, Journal of Antimicrobial Chemotherapy,
(2003), 51, 373378
William D. and Lemke, T. 2002, Foye's Principles of Medicinal Chemistry, 5th edn, Lippincott, Williams
and Wilkins, Baltimore.
Witkowski, G., Lode, H., Hffken, G. and Koeppe, P., Pharmacokinetic studies of amoxycillin, potassium
clavulanate and their combination, European Journal of Clinical Microbiology (1982), 1, 233237
Wright. G. Bacterial Resistance to antibiotics: Enzyme degradation and modification. Advanced Drug