Abstract

Amoxycillin is a β-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 β-lactam antibiotics is via production of β-

lactamases. These β-lactamases act to destroy the β-lactam ring of

amoxycillin, rendering it inert. In order to combat this growing problem,

amoxycillin is often combined with clavulanic acid, a β-lactamase

inhibitor. This agent inhibits the action of β-lactamases, protecting the

amoxycillin and allowing it to destroy the bacteria. The following article

reviews the chemistry, pharmacodynamics, pharmacokinetics and

metabolism of this highly effective combination.

Introduction

Amoxycillin is a β-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 β-lactam ring is required for

antibacterial action; however it is susceptible to inactivation by β-lactamases. Β-

lactamase is produced by bacteria and acts to destroy the β-lactam ring of amoxycillin.

The problem of bacterial resistance to β-lactams is increasing in prevalence due to

widespread exposure over the last 50 years and the continuous evolution of β-lactamases

(Miller et al, 2001).

The use of β-lactamase inhibitors in combination with β-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 β-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 β-lactam/ β-lactamase inhibitor combinations has

been the subject of much research. In the future more research is required to develop

new β-lactamase inhibitors capable of combating the continually changing epidemiology

of β-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

AmoxycillinThe basic structure of penicillins consists of a thiazolidine ring (A) connected to a β-

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 β-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 β-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 β-lactam ring from β-lactamases, which are produced by resistant bacteria

and render the antibiotic inactive (Beleh, M., 2006).

Mechanism of ActionAmoxycillin'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 β-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])

StabilityAmoxycillin 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 15–20 hours in solution with a pH of 2 at 35°C (AHFS

Drug Information [online]).

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

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

bacteria. β-lactamases attack the β-lactam ring at the carbonyl position (see figure 4.)

opening the β-lactam and thus inactivating amoxycillin (Foye’s Med. Chem, 2002). To

avoid this amoxycillin is prepared in combination products that include the β-lactamase

inhibitor, clavulanic acid. This inhibition is irreversible and is known as “suicide

inhibition”. This allows amoxycillin to produce antibacterial effects.

Clavulanic AcidClavulanic 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 ActionClavulanic acid is believed to acylate the active site serine by mimicking the normal

substrate (Foye’s Med. Chem, 2002). While hydrolysis can still occur with some β-

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 β-lactamase producing

strains is markedly enhanced.

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

Pharmacodynamics of Amoxycillin and Clavulanic Acid

AmoxycillinMicrobiological 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 (post-

antibacterial 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 β-

lactamases. These β-lactamases are produced by the targeted organism and act to cleave

the β-lactam ring of amoxycillin. Specifically, an active site serine on the β-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 AcidClavulanic acid is a β-Lactamase inhibitor which possesses little intrinsic antibacterial

activity. It inhibits the action of β-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 β-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 β-lactamases. For example the MIC

of amoxycillin against Haemophilus influenzae is >64mg/L. This bacterium produces β-

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 MIC’s of many strains

becoming too high for effective treatment with current combinations, new,

pharmacodynamically and pharmacokinetically enhanced formulations have been

developed. For example, a new paediatric formulation uses a 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 MIC’s can be effectively treated,

as the T>MIC remains above 40% for these bacteria. (Navarro, 2005)

Pharmacokinetics of Amoxycillin and Clavulanic Acid

PharmacokineticsClavulanic 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 β-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 1-

week washout period) crossover Phase I bioequivalence studies, with following Co-

amoxiclavTM (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 concentration–time 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 (A1-

A4 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 2.04±1.01 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 AUCt’s of amoxycillin plotted versus the mean AUCt’s 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 β-lactams, such as amoxycillin, the

unbound serum concentration of the drug exceeding the MIC of the causative pathogen

for 40–50% 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

AmoxycillinThe amide bond of the β-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 β-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 β-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 acidClavulanic 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-4-

hydroxy-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 β-lactamase producing bacteria after nearly 20 years

of clinical use. Different classes of β-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 β-lactam/β-

lactamase inhibitor products. This is due to the fact that the prevalence and continuing

evolution of bacteria producing β-lactamases is also increasing. While several

compounds have shown promise in inhibition of both class A and C β-lactamases, none

of them have yet made it to clinical trials. Hence, second-generation β-lactamase

inhibitors, which inhibit both class A and C β-lactamases, and are combined with an

appropriate β-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.

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