Cell wall synthesis inhibitors

Cell Wall Synthesis Inhibitors

•       Penicillins

•       Cephalosporins

•       Monobactams

•       Carbapenems

•       Glycopeptides

•       Other Cell Wall- or Membrane-Active Agents

BETA-LACTAM ANTIBIOTICS

(inhibitors of cell wall synthesis)

Their structure contains a beta-lactam ring.

The major subdivisions are:

penicillins whose official names usually include or end in “cillin”

b.       cephalosporins which are recognized by the inclusion of “cef” or “ceph” in their official names.

c.       carbapenems (e.g. meropenem, imipenem)

d.       monobactams (e.g. aztreonam)

e.       beta-lactamase inhibitors (e.g. clavulanic acid, sulbactam).

Chemical Structure

Penicillins

•               In 1928, Alexander Fleming was researching vaccines and noticed a culture of staphlococci had undergone lysis from contamination with a mold

•               Fleming finally isolated the mold, Penicillium notatum, and found that the fluid beneath it possessed antibacterial properties

•               Basic structure consists of thiazolidine ring connected to b-lactam ring, to which is attached a side chain

Structure of penicillins and products of their enzymatic hydrolysis:

Chemical structure, major variants

Classification of penicillins

Penicillins are subclassed based on

                chemical structure (eg, penicillins, monobactams, and  carbapenems),

                spectrum (narrow, broad, or extended)

                source (natural, semisynthetic, or synthetic) and          

susceptibility    to β-lactamase destruction.

Classes by Spectrum

Narrow-spectrum β-Lactamase-Sensitive Penicillins

                Penicillin G (benzylpenicillin); penicillin V [phenoxymethyl-penicillin] and phenethicillin.

                These are active against many gram-positive and a limited number of gram-negative bacteria, as well as anaerobic organisms, but they are susceptible to β-lactamase (penicillinase) hydrolysis.

Narrow-spectrum β-Lactamase-Resistant Penicillins

                This group, through substitution on the penicillin nucleus (6-aminopenicillanic acid), is refractory to a greater or lesser degree to the effects of various β-lactamase enzymes produced by resistant gram-positive organisms, particularly Staphylococcus aureus.

                However, penicillins in this class are not as active against many gram-positive bacteria as penicillin G and are inactive against almost all gram-negative bacteria.

                Acid-stable members of this group may be given orally and include isoxazolyl penicillins, such as oxacillin, cloxacillin, dicloxacillin, and flucloxacillin.

                Methicillin and nafcillin are available as parenteral preparations.

                Temo-cillin is a semisynthetic penicillin that is β-lactamase stable but also active against nearly all isolates of gram-negative bacteria except Pseudomonas spp.

Broad-spectrum β-Lactamase-Sensitive Penicillins

                Penicillins in this class are derived semisynthetically and are active against many gram-positive and gram-negative bacteria. However, they are readily destroyed by the β-lactamases (produced by many bacteria).

                Many members of the group are acid stable and are administered either PO or parenterally.

                Of those used in veterinary medicine, aminopenicillins, eg, ampicillin and amoxicillin, are the best known. Several ampicillin precursors that are more completely absorbed from the GI tract also belong to this class (eg, hetacillin, pivampicillin, talampicillin). Mecillinam is less active than ampicillin against gram-positive bacteria but is highly active against many intestinal organisms (except Proteus spp) that do not produce β-lactamases.

Broad-spectrum β-Lactamase-Sensitive Penicillins with Extended Spectra

                Several semisynthetic broad-spectrum penicillins are also active against Pseudomonas aeruginosa, certain Proteus spp, and even strains of Klebsiella, Shigella, and Enterobacter spp in certain cases. Examples of this class include carboxypenicillins (carbenicillin, its acid-stable indanyl ester, and ticarcillin), ureido-penicillins (azlocillin and mezlocillin), and piperazine penicillins (piperacillin).

β-Lactamase-Protected Broad-Spectrum Penicillins

                Several naturally occurring and semisynthetic compounds can inhibit many of the β-lactamase enzymes produced by penicillin-resistant bacteria.

                When used in combination with broad-spectrum penicillins, there is a notable synergistic effect because the active penicillin is protected from enzymatic hydrolysis—and thus is fully active against a wide variety of previously resistant bacteria.

                Examples of this chemotherapeutic approach include clavulanate-potentiated amoxicillin and ticarcillin as well as sulbactam-potentiated ampicillin and tazobactam-potentiated piperacillin.

Carbapenems

                Imipenem and meropenem are among the most active drugs against a wide variety of bacteria.

                Imipenem is derived from a compound produced by Streptomyces cattleya.

                Aztreonam is a related (monobactam) compound but differs from other β-lactams in that it has a second ring that is not fused to the β-lactam ring.

Antibacterial Spectrum

Natural penicillins

Penicillin G (Benzylpenicillin)

•       Gram +/- cocci, Gram + bacilli, spirochetes, anaerobes

•       Susceptible to inactivation by b-lactamase

Penicillin V

•       Similar spectrum

•       More acid stable than Pen G

•       Higher minimum inhibitory concentration

Bacteria Susceptible to Penicillin

Strep pneumonia - Major cause of bacterial pneumonia in all ages

Gonorrhea - Neisseria gonorrhea and meningitidis

Syphilis - Treponema pallidum

Antistaphylococcal penicillins

Methicillin

Nafcillin

Oxacillin

Dicloxacillin

All are penicillinase-resistant penicillins used in penicillinase-producing staphylococci

Methicillin-resistant staph aureus

Extended spectrum penicillins

Ampicillin

Amoxicillin

Activity against haemophilus influenza, proteus mirabilis, E. coli, and neisseria species

Resistance due to plasmid mediated penicillinase

May use clavulanic acid or sulbactam to extend the antibacterial activity

Antipseudomonal penicillins

Carbenicillin

Ticarcillin

Piperacillin

Azlocillin

Mezlocillin

Effective against pseudomonas aeroginosa and other gram negatives

General Properties

The penicillins are somewhat unstable, being sensitive to heat, light, extremes in pH, heavy metals, and oxidizing and reducing agents. Also, they often deteriorate in aqueous solution and require reconstitution with a diluent just before injection. Penicillins are poorly soluble, weak organic acids that are administered parenterally either as suspensions in water or oil, or as water-soluble salts. For example, sodium or potassium salts of penicillin G are highly water soluble and are absorbed rapidly from injection sites, whereas organic esters in microsuspension such as procaine penicillin G or benzathine penicillin G are gradually absorbed over 1–3 (or even more) days, respectively. The trihydrate forms of the semisynthetic penicillins have greater aqueous solubility than the parent compounds and are usually preferred for both parenteral and oral use.

Penicillins contain a β-lactam nucleus that when cleaved by a β-lactamase enzyme (penicillinase) produces penicilloic acid derivatives that are inactive but may act as the antigenic determinants for penicillin hypersensitivity. Modification of the 6-aminopenicillanic acid nucleus, either by biosynthetic or semisynthetic means, has produced the array of penicillins used clinically. These differ in their antibacterial spectra, pharmacokinetic characteristics, and susceptibility to microbial enzymatic degradation.

Penicillin G and its oral congeners (eg, penicillin V) are active against both aerobic and anaerobic gram-positive bacteria and, with a few exceptions (Haemophilus and Neisseria spp and strains of Bacteroides other than B fragilis), are inactive against gram-negative organisms at usual concentrations. Organisms usually sensitive in vitro to penicillin G include streptococci, penicillin-sensitive staphylococci, Arcanobacterium pyogenes, Clostridium spp, Erysipelothrix rhusiopathiae, Actinomyces bovis, Leptospira canicola, Bacillus anthracis, Fusiformis nodosus, and Nocardia spp.

The semisynthetic β-lactamase-resistant penicillins, such as oxacillin, cloxacillin, floxacillin, and nafcillin, have spectra similar to those noted above (although often at higher MIC) but also include many of the β-lactamase-producing strains of staphylococci (especially S aureus and S epidermidis).

A large number of gram-positive and gram-negative bacteria (but not β-lactamase-producing strains) are sensitive to the semisynthetic broad-spectrum penicillins (ampicillin and amoxicillin). Susceptible genera include Staphylococcus, Streptococcus, Arcanobacterium, Clostridium, Escherichia, Klebsiella, Shigella, Salmonella, Proteus, and Pasteurella. While bacterial resistance is widespread, the combination of β-lactamase inhibitors and broad-spectrum penicillins markedly enhances the spectrum and efficacy against both gram-positive and gram-negative pathogens. Clavulanate-potentiated amoxicillin is an excellent example of such a synergistic association.

The anti-Pseudomonas and other extended-spectrum penicillins are active against most of the usual penicillin-sensitive bacteria. They often have a degree of β-lactamase resistance and are usually active against one or more characteristic penicillin-resistant organisms. Yet, as a class, they remain susceptible to destruction by β-lactamases. Examples include the use of carbenicillin, ticarcillin, and piperacillin against Pseudomonas aeruginosa and several Proteus strains, and the use of piperacillin against Pseudomonas aeruginosa, several Shigella and Proteus strains, and some Citrobacter and Enterobacter spp. Streptococcus faecalis is often resistant to these new extended-spectrum penicillins. Imipenem and meropenem are relatively resistant to β-lactamase destruction. Their spectrum includes a wide variety of aerobic and anaerobic microorganisms, including most strains of Pseudomonas, streptococci, enterococci, staphylococci, and Listeria. Anaerobes, including Bacteroides fragilis, are highly susceptible.

Difference between Gram +ve and –ve bacteria in their cell wall structure

Cell envelope of a gram-negative bacterium

The cell wall completely surrounds the cytoplasmic membrane, maintains cell shape and integrity, and prevents cell lysis from high osmotic pressure.

The cell wall is composed of a complex cross-linked polymer of polysaccharides and polypeptides, peptidoglycan (murein, mucopeptide).

                The polysaccharide contains alternating amino sugars, N-acetylglucosamine (NAGA), and N-acetylmuramic acid (NAMA).

                A five-amino-acid peptide is linked to the N-acetyl muramic acid sugar. This peptide terminates in D-alanyl-D-alanine.

Penicillin-binding protein (PBP, an enzyme transpeptidases / endopeptidases / carboxypeptidases)) removes the terminal alanine in the process of forming a cross-link with a nearby peptide.

                Cross-links give the cell wall its structural rigidity.

                β-lactam antibiotics covalently bind to the active site of PBPs. This inhibits the transpeptidation reaction, halting peptidoglycan synthesis, and the cell dies.

                β-lactams kill bacterial cells only when they are actively growing and synthesizing cell wall.

Mechanism of action

ß-lactam antibiotics are bactericidal drugs. They inhibit cell wall synthesis by the following steps:

 1- Binding of the drug to specific receptors (penicillin-binding proteins, PBPs)

 2- Inhibition of transpeptidases that act to cross-link linear peptidoglycan chains that form part of the cell wall.

3- Activation of autolytic enzymes that cause lyses of the bacterial cell wall.

•       Interferes with last step of bacterial cell wall synthesis, causing cell lysis

•       Bactericidal

•       Only effective against rapidly growing organisms that synthesize a peptidoglycan cell wall

•       Inactive against mycobacteria, fungi, viruses

Mechanism of Action way of being bactericide

•       Penicillin binding proteins

–      Enzymes involved in forming cross linkages between peptidoglycan chains inactivated by penicillin

•       Inhibition of transpeptidase

–      Hinders last step in formation of cross links needed for cell wall integrity

•       Autolysins

–      Normally degrade cell wall, but penicillin prevents new synthesis

The transpeptidation reaction in Staphylococcus aureus that is inhibited by β-lactam antibiotics. The cell wall of gram-positive bacteria is made up of long peptidoglycan polymer chains consisting of the alternating aminohexoses N-acetylglucosamine (G) and N-acetylmuramic acid (M) with pentapeptide side chains linked (in S aureus) by pentaglycine bridges. The exact composition of the side chains varies among species. The diagram illustrates small segments of two such polymer chains and their amino acid side chains. These linear polymers must be cross-linked by transpeptidation of the side chains at the points indicated by the asterisk to achieve the strength necessary for cell viability.

The biosynthesis of cell wall peptidoglycan, showing the sites of action of five antibiotics (shaded bars; 1 = fosfomycin, 2 = cycloserine, 3 = bacitracin, 4 = vancomycin, 5 = -lactam antibiotics). Bactoprenol (BP) is the lipid membrane carrier that transports building blocks across the cytoplasmic membrane; M, N-acetylmuramic acid; Glc, glucose; NAcGlc or G, N-acetylglucosamine.

Bacterial Resistance

1. β-lactamase activity

•          Hydrolyzes cyclic amide bond of b-lactam ring

•          Usually acquired by transfer of plasmids

2. Decreased permeability to drug
3. Altered penicillin binding proteins

•          May require greater antibiotic concentrations

4. Efflux

Pharmacokinetic Features

Absorption

                Most penicillins in aqueous solution are rapidly absorbed from parenteral sites.

                Absorption is delayed when the inorganic penicillin salts are suspended in vegetable oil vehicles or when the sparingly soluble repository organic salts (eg, procaine penicillin G and benzathine penicillin G) are administered parenterally.

                Although prolonged absorption results in longer persistence of plasma and tissue drug concentrations, peak concentrations may not be sufficiently high to be effective against organisms unless MIC are low.

                The penicillin G repositol salts should never be injected IV.

                Only selected penicillins are acid stable and can be administered PO at standard doses. Absorption from the upper GI tract differs markedly in amount and rate among the various penicillins. Penicillin V must be given at high oral doses.

                The aminopenicillins are orally bioavailable, although food impairs the absorption of ampicillin.

                The indanyl form of carbenicillin is orally bioavailable, but effective concentrations are likely to be achieved only in the urine.

                Serum concentrations of penicillins generally peak within 2 hr of PO administration. Penicillins may also be absorbed after intrauterine infusion.

Distribution                                 

                After absorption, penicillins are widely distributed in body fluids and tissues.

                The volume of distribution tends to reflect extracellular compartmentalization, although some penicillins penetrate into tissues quite well.

                Potentially therapeutic concentrations of the various penicillins are generally found in the liver, bile, kidneys, intestines, muscle, and lungs, but only very low concentrations are found in poorly perfused areas such as the cornea, bronchial secretions, cartilage, and bone.

                The diethylamino salt of penicillin G produces particularly high concentrations in pulmonary tissue.

                The penicillins usually do not readily cross the normal blood-brain, placental, mammary, or prostatic barriers unless massive doses are given or inflammation is present.

                Selected penicillins are able to penetrate nonchronic abscesses and pleural, peritoneal, or synovial fluids.

                Penicillins are reversibly and loosely bound to plasma proteins. The extent of this binding varies with particular penicillins and their concentration, eg, ampicillin is usually ∼20% bound, and cloxacillin may be ∼80% bound.

                Pregnancy increases the volume of distribution, which has the effect of lowering the concentration of drug produced by a given dose.

Biotransformation

                Penicillins are generally excreted unchanged, but fractions of a given dose may undergo metabolic transformations by unknown mechanisms (usually <20% metabolized).

                Penicilloic acid derivatives that are formed tend to be allergenic.

Excretion        

                Most (60–90%) of a parenterally administered penicillin is eliminated in the urine within a short time (eg, up to 90% of penicillin G within 6 hr), which results in high concentrations in urine.

                About 20% of renal excretion occurs by glomerular filtration and ∼80% by tubular secretion—a process that may be deliberately inhibited (to prolong effective concentrations in the body) by probenecid and other weak organic acids.

                Anuria may increase the half-life of penicillin G (normally ∼30 min) to 10 hr.

                The biliary route also may be a major excretory pathway for the broad-spectrum semisynthetic penicillins. Clearance is considerably lower in neonates than in adults.

                Penicillins are also eliminated in milk, although often only in trace amounts in the normal udder, and may persist for up to 90 hr. Penicillin residues in milk also have been found after intrauterine infusion.

Elimination, Distribution, and Clearance of Penicillins
Penicillin	Species	Elimination Half-life (min)	Volume of Distribution (mL/kg)	Clearance (mL/kg/min)
Penicillin G	Dogs	30	156	3.6
	Horses	38	301	5.5
Ampicillin	Dogs	48	270	3.9
Amoxicillin	Cattle	84	493	4.0
Ticarcillin	Dogs	48	347	4.9
Carbenicillin	Cattle	122	330	5.5

One std. Unit of penicillin is defined as the amount of antibacterial activity present in 0.6 µgm of pure crystalline standard sodium penicillin G.

1 mg = 1667 oxford units.

The dosage of semi-synthetic penicillins is expressed in mg/kg.

Specific Indications for Penicillins

•       Narrow-spectrum bactericidal

•       Most Gm+ cocci and rods and anaerobes

•       Intravenous: Pen G (potassium or sodium)

•       Intramuscular: Benzathine or procaine Pen G

•       Oral: Pen V

•       Pen V, G well distributed in soft tissues

•       Bone level is fraction of plasma concentration

•       Excreted primarily via kidneys

Special Clinical Concerns

Adverse Effects and Toxicity:   Organ toxicity is rare.

                Hypersensitivity reactions (particularly in cattle) include skin reactions, angioedema, drug fever, serum sickness, vasculitis, eosinophilia, and anaphylaxis. Cross-sensitivity among penicillins is well recognized.

                Intrathecal administration may result in convulsions.

                Guinea pigs, chinchillas, birds, snakes, and turtles are sensitive to procaine penicillin.

                The use of broad-spectrum penicillins may lead to superinfection, and GI disturbances may occur after PO administration of ampicillin.

                Potassium penicillin G should be administered IV with some caution, especially if hyperkalemia is present.

                The sodium salt of penicillin G may also contribute to the sodium load in congestive heart failure.

Adverse Reactions

•       Hypersensitivity- 1-10% patients treated

–      Penicilloic acid- hapten for immune reaction

–      Urticaria to angioedema to anaphylaxis

–      b-lactam cross reactivity

•       Diarrhea- disruption of flora- ampicillin

•       Nephritis- methicillin

•       Neurotoxicity- intrathecal or seizure disorders

•       Platelet dysfunction- carbenicillin, ticarcillin

•       Cation toxicity- watch sodium (congenstive heart failure) and potassium (cardiac toxicity esp in renal failure pts)

•       Skin rashes-ampicillin and amoxicillin 

Allergic Reactions

•       Acute (< 30 min)

–      Urticaria, angioedema, bronchoconstriction, shock

•       Accelerated (30 min-48 hrs)

–      Urticaria, pruritis, wheezing, mild laryngeal edema, local inflammatory reactions

•       Delayed (> 2 days)

–      Skin rash

–      Oral glossitis, flurred tongue, black and brown tongue, cheilosis, severe stomatitis with loss buccal mucosa

•       Mild: Diphenhydramine 25-50 mg IV/IM/PO

•       Severe: Epinephrine 0.03-0.05 mg

•       Skin tests: benzylpenicilloyl-polylysine

Interactions

                Tubular secretion is delayed in the presence of selected organic ions, including salicylates, phenylbutazone, sulfonamides, and other weak acids.

                Gut-active penicillins potentiate the action of anti-coagulants by depressing vitamin K production by gut flora.

                 Absorption of ampicillin is impaired by the presence of food.

                β-lactams in general interact chemically with the aminoglycosides and should not be mixed in vitro.

                Ampicillin and penicillin G are incompatible with many other drugs and solutions and should not be mixed.

Penicillins and Aminoglycosides

•       Synergistic

•       Inactivate each other if placed in same IV fluid bag because of positively charged aminoglycosides and negative penicillins

β-lactamase Inhibitors

•       Clavulanic acid: product of streptomyces clavuligerus

•       Irreversibly binds b-lactamases and inactivates them

•       Augmentin: amoxicillin + clavulanic acid

•       Timentin: ticarcillin + clavulanic acid

•       Unasyn: ampicillin + sulbactam