Living Textbook MC610

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Stage III Inhibitors:

β-LACTAMS

A lactam is an internal amide, just like a lactone is an internal ester. A β-lactams indicates that the amine is in the β-position in relation to the carboxylic acid. All β-lactams will have similar mechanisms of action and many general properties.

PENICILLINS

They are the first group of antibiotics discovered.

Source:

Fermentation of Penicillium sp., which would lead to a mixture of compounds, including Penicillin G, V, O and S. We can obtain exclusively Penicillin G by the addition of phenyl acetic acid.

General Properties:

  1. Orally active, can be absorbed from the GIT.
  2. Readily destructed in neutral or basic solutions. Opening of the lactam ring occurs, resulting in an inactive compound.
  3. Acid degradation takes place, where two third of the dose is destroyed in the stomach.
  4. Low toxicity, only major problem is immunological reaction in some patients.

Spectrum :

Against Gram positive such as Staphylcoccus sp., gonococcus sp. and pneumococcus sp.

  1. Can not cross Gram negative bacterial cell walls.
  2. Destroyed by "Penicillinase"enzyme found in Gram negative bacteria.

Useful in such cases such as scarlet fever, endocarditis, pneumonia, sepsis, meningitis and anthrax.

After a few years of use (by 1948), resistance developed in many cases, reaching 50% in hospital patients, which reached 75% later on.

Scientists tried to find other agents, that are:

1. Broad spectrum in action, 2. Orally active and 3. Builds up less or no resistance.

The first attempts were to add other substrates to the fermentation media and thus obtain other Penicillins with the desired properties. The only one that is still in use from these earlier attempts is Penicillin V.

No dramatic improvement, except improved acid stability.

Long acting Penicillins were introduced by using Penicillin G salts, procaine and benzathine, which formed a depot in the body and prolonged therapeutic blood levels were thus accomplished.

Then in 1957 came the first attempts at total synthesis of Penicillins which turned out to be very expensive and inefficient and thus unpractical.

Then a huge discovery in 1959, an enzyme that can chop off the acyl side chain was found in resistant bacteria. The enzyme is an "acylase", and resulted in the formation of 6-aminopenicillanic acid (6-APA), which is an inactive species.

Now we can add any side chains we need by using an acid chloride or acid anhydride, or using the carboxylic acid either in the presence of an amidase, pH = 5 or DCCI as a water scavenger (semi-synthetic).

Scientists were then able to examine what problems may limit Penicillins and improve on them.

  1. Spectrum : Why are older Penicillins inactive against Gram negative bacteria?
    1. Cell wall permeability.
    2. Solution : Add polar groups.

    3. Penicillinase activity, both an acylase and a β-lactamase.

    4. Solution : Add bulky groups as close as possible to the ring, leading to a steric hinderness and the enzyme is unable to break the β-lactam.

  2. Why are they acid labile ?
  3. Solution : Add electron withdrawing groups at the R-position.

  4. Why do some organisms become resistant ?
  5. Mainly due to β-lactamase production, that is enhanced by mutations, induction and natural selection, and by a change in cell wall permeability.

    Solution : Similar to Number 1.

So scientists started to produce a wide variety of Penicillins with varying side chains and from these came the agents that are used clinically today.

But you have to make sure that these modifications do not alter activity, so we look at:

Structure Activity Relationship :

1-position, must have a nitrogen.

2-position, the carboxylic acid is a must. If changed to an alcohol/ester, it will be inactive.

3-position, any change will lower activity.

4-position, if the sulfur is oxidized to the sulfone/sulfoxide, it gives it better acid stability, but becomes less active.

5-position, no substitutions allowed.

7-position, must have the carbonyl

Now the 6-position, specifically the R group:

  • If add an electron withdrawing group, then the amide oxygen will be less nucleophillic and will thus give the compound better acid stability.
  • If a bulky group is added close to the ring, then it makes the drug better resistant to β-lactamases, since it can not enter the active site of the enzyme.
  • If a polar group is added, its spectrum becomes broader, since it can pass through the porins in the Gram negative bacteria cell walls.
  • Mechanism of Action:

    Inhibits stage III and thus prevents cross-linking of peptidoglycan, based on its structural similarity to the Ala-Ala terminal of the peptidoglycan.

    In 3-D, Penicillins resemble the transition state, thus are transition state inhibitors.

    Mechanistically, it involves a nucleophillic attack by the enzyme leading to the opening of the β-lactam ring and the formation of an enzyme-penicillin intermediate. Tautomerization gives the intermediate stability, thus leading to enzyme inhibition.

    In addtion, steric hinderness prevents the nucleophile (Glycine in the biosynthetic pathway) from entering the active site and releasing the enzyme.

    Penicillins form a stable complex, as determined by the enzymatic equation. The faster the formation of the EI* (=K3/K1) and the slower the degradation step (K4), the better the inhibition.

    However it is not that simple. It turns out that Penicillins bind to different membrane bound Penicillin Binding Proteins (PBP) that have different functions during stage III and penicilins differ in their affinity to the different PBPs.

    PBP A and B are the most important, leading to the synthesis and elongation of the peptidoglycan. They have a transpeptidase and transglycosidase activity, that if inhibited will cause cell lysis.

    PBP C is a carboxypeptidase that removes the last D-Ala to limit cross linking.

    Each β-lactam binds to a specific PBP, thus each has its own activity. However, all have a similar active site containing a serine residue.

    Resistance

    There are several methods of resistance. The most important and abundant is teh production of β-lactamases that inactivate these drugs by attacking and breaking the β-lactam ring. There are a number of β-lactamases that differ from one bacterial species to the other. One difference is the presence of a serine or a metal ion in the active site. Some of these enzymes are more specific to one class or another, such as penicillinases, cephalosporinases and carbapenemases.

    Another important mechanism of resistance involves mutations to the PBP. For example, methicillin-resisatnt staph (MRSA) has a mutated type of PBP2a that β-lactams show low binding capability.

    Other Considerations :

    1. Penicillins are all acids, with a pKa around 2.5 to 3. They are crystalline and should be protected from moisture. If it is stored under dry conditions, it will be stable for years.
    2. They have an unpleasant taste.
    3. They are not suitable to formulate as a free acid, so used as sodium or potassium salts.
    4. Generally safe in pregnancy and during lactation. It will pass into the milk, so only precaution is to watch out for anaphylactic shock and diarrhea.
    5. Differ in their protein binding capability, between 20 and 90% protein bound. This leads to a change in the free drug available in the bloodstream but not in their duration of action.
    6. Immunological Reaction; An anaphylactic shock can occur in less than 0.1% of the population, or a rash in 5 - 7%. This results from the reaction of the β-lactam ring with a terminal amine on a lysine in a polypeptide, leading to the formation of an allergen. Antibodies are formed the first time the patient is exposed to the antibiotic, and the reaction will be severe in consequent times. If the preparation is not well purified, then residual proteins will cause the reaction to be more severe. First line of treatment is the use of Adrenaline to revive the blood pressue and relieve the bronchoconstriction.
    7. Several drug interactions have been reported, such as oral contraceptives and the antigout, probenicid. This later compound will compete with Penicillins for active tubular sites of active excretion, resulting in higher blood levels and may lead it to reach toxic levels.

    Examples of Clinically Useful Penicillins

    The first penicillin used was Penicillin G. It is used in units, where according to the USP one unit is equal to the antibiotic activity present in 0.6 µg. It is effective against only Gram positive bacteria, is acid labile and can be destroyed by β-lactamases. It is used as its sodium or potassium salt in parentral preparations. Penicillin G is a cheap and effective agent that lacks toxicity and is often employed in upper and lower RTI and Genitourinary infections. The procaine salt is used in IM injections and can be given only once a day, while the benzathine salt is give a more extended action.

    Penicillin V has the advantage of being acid stable due to the electron withdrawing side chain. It is thus used in oral preparations. It has the same spectra as Penicillin G, but is less potent.

    The bulky groups in Methicillin and Nafcillin make them less susceptible to attacks by β-lactamases. The former is less potent and has a narrow spectrum compared to Penicillin G but effective against β-lactamase producing Staphylococcus aureus. The latter is more stable in the acidic environment of the stomach.

    Isoxazole penicillins are both β-lactamase resistant and acid stable, thus used in oral preparations. The chlorine moiety leads to an increase in both absorption and protein binding, which can be reflected in the dose differential. While Oxacillin is given as 500 to 1000 mg oral capsules every 4 hours, Cloxacillin's regimen is usually 250-500 mg every 6 hours, and Dicloxacillin's is 125-250 mg every 6 hours.

    Amino penicillins are both acid stable and have a wide spectrum of activity due to the polar side chain. They are however still susceptible to β-lactamases. Ampicillin is less active than penicillin G, but is effective against Gram negative bacteria such as E. Coli and hemophillus Influenza. It can form a Zwitter ion so its oral absorption is average (30-55 %), and should not be administered with meals. The unabsorbed drug may affect the natural flora and leads to the relatively high incidences of diarrhea. It is usually administered every 6 hours.

    Amoxicillin on the other hand is much better absorbed (when compared to Ampicillin). It can be administered every 8 to 12 hours

    Antipseudomonal Penicillins share their wide spectrum of activity and show very good activity against pseudomonal infections. They are also stable to acidic medium due to the polar side chain. Carbenicillin is decarboxylated in the stomach and is thus not suitable for oral administration, but its indanyl ester can be used orally. Ticarcillin has a better pharmacokinetic profile and is a more effective agent than Carbenicillin. Other examples include Mezlocillin and Piperacillin, which is the most active agent in this group, probably due to the urea-like substitution that mimics the cell wall, causing better binding to PBPs.

    Clinical Applications:

    General: Bactericidal, older agents with a narrow spectrum, which improved with newer agents.

    Penicillin G and V:

    Coverage includes Gram positive bacteria: Staphylococcus and Streptococcus and some Gram negative bacteria: Neisseria gonorrhoeae and meningitidis.  Most Staph are resistant.

    Indications and Uses: Various infections caused by non-beta-lactamase producing susceptible organisms.

    Nafcillin, Oxacillin and Dicloxacillin:

    Coverage same as penicillin G but has activity against some beta-lactamase producing agents.  Not for MRSA or enterococcus.

    Indications and Uses: Various Gram positive infections, including some that are caused by beta-lactamase producing organisms.

    Ampicillin and Amoxicillin:

    Coverage same as Penicillin G but add some non-beta-lactamase producing Gram negative organisms, such as E. coli, H. influenzae, and proteus mirabilis.  Gram positive Enterococcos faecalis is susceptible to Ampicillin.

    Adding a Beta-lactamase inhibitor (clavulanic acid or sulbactam) extends the coverage to include beta-lactamase producing Staph and H. influenzae, but not E. coli.  It also includes Acinetobacter and Klebisella.

    Indications and Uses: Variety of infections caused by susceptible organisms.  They are useful in CAP, but only in preparations containing beta-lactamase inhibitors (such as Augmentin).

    Piperacillin:

    Coverage same as ampicillin but add non-beta-lactamase producing pseudomonas

    Adding a Beta-lactamase inhibitor extends coverage to include beta-lactamase producing pseudomonas, Acinetobacter and Klebisella.

    Indications and Uses: Usually for infections caused by Pseudomonas aeruginosa.