Strategies to overcome β-lactamase sensitivity, Mechanism based inhibition of Beta-lactamases, Carbapenems

1. Beta-LACTAMASE INHIBITORS
Dr. Shilpa S. Harak

2. Strategies to overcome β-lactamase sensitivity
 Combination of
β-lactamase inhibitor with β-lactamase–sensitive penicillin
in infections caused by β-lactamase–producing bacterial strains
Result - failed
 Synergy against resistant strains,
Combinations consisting of a
β- lactamase–resistant penicillin (e.g., methicillin or oxacillin) as a competitive inhibitor
and
β -lactamase– sensitive penicillin (e.g., ampicillin or carbenicillin) to kill the organisms,
met with limited success.

3. Factors responsible for synergy combination’s failure
(a) the failure of most lipophilic penicillinase-resistant penicillins to penetrate the cell
envelope of Gram-negative bacilli in effective concentrations,
(b) the reversible binding of penicillinase-resistant penicillin to β-lactamase, requiring high
concentrations to prevent substrate binding and hydrolysis, and
(c) the induction of β-lactamases by some penicillinase-resistant penicillin.
NEW HOPE
 Discovery of mechanism based inhibitor clavulanic acid, which causes potent and
progressive inactivation of β-lactamases, has created renewed interest in β-lactam
combination therapy.

4. Classification
 Class I
β-lactamase inhibitors with a β-lactam core, have a heteroatom leaving
group at position 1 (e.g., clavulanic acid and sulbactam)
 Class II inhibitors
β-lactamase inhibitors without a β-lactam core: do not have a heteroatom
leaving group at position 1 (e.g., the carbapenems).
 Mechanism-based inhibitors react with the enzyme in the same way that the
substrate does,
 Unlike competitive inhibitors, which bind reversibly to the enzyme they inhibit.
 With the β-lactamases, an acyl-enzyme intermediate is formed by reaction of
the β-lactam with an active-site serine hydroxyl group of the enzyme.

5. Two classes of Beta-lactamase inhibitors:
 For normal substrates, the acyl-enzyme intermediate readily undergoes
hydrolysis, destroying the substrate and freeing the enzyme to attack
more substrate
 The acyl-enzyme intermediate formed when a mechanism-based inhibitor
is attacked by the enzyme is diverted by tautomerism to a more stable
imine form that hydrolyzes more slowly to eventually free the enzyme
(transient inhibition), or for a class I inhibitor, a second group on the
enzyme may be attacked to inactivate it.
 Because these inhibitors are also substrates for the enzymes that they
inactivate, they are sometimes referred to as “suicide substrates.”

6. Two classes of Beta-lactamase inhibitors:
 Because class I inhibitors cause prolonged inactivation of certain β-
lactamases,
 they are particularly useful in combination with extended-spectrum,
 β-lactamase–sensitive penicillins to treat infections caused by β -lactamase–
producing bacteria.
 Three such inhibitors, clavulanic acid, sulbactam, and tazobactam, are
currently marketed in the United States for this purpose.
 A class II inhibitor, the carbapenem derivative imipenem, has potent
antibacterial activity in addition to its ability to cause transient inhibition of
some β -lactamases.
 Certain antibacterial cephalosporins with a leaving group at the C-3 position
can cause transient inhibition of β - lactamases by forming stabilized acyl-
enzyme intermediates.

7. Class I inhibitors - β -lactamases inactivation
The relative susceptibilities is related to the molecular properties of the
enzymes.
 Group A - serine enzymes:
 Narrow specificities (e.g., penicillinases or cephalosporinases)
 Broad specificities (i.e., general β -lactamases),
 inactivated by class I inhibitors.
 Group B
 Zn2-requiring metallo-β-lactamases with broad substrate specificities
 are not inactivated by class I inhibitors.
 Group C
 Chromosomally encoded serine β-lactamases with specificity for
cephalosporin
 resistant to inactivation by class I inhibitors.

8. Mechanism based
inhibition of Beta-
lactamases

9. Class I inhibitors

10. Clavulanic acid
 Clavulanic acid is an antibiotic isolated from Streptomyces
clavuligeris.
 Structurally, it is a 1-oxopenam lacking the 6-acylamino side chain
of penicillins but possessing a 2-hydroxyethylidene moiety at C-2.
 Clavulanic acid exhibits very weak antibacterial activity,
comparable with that of 6-APA and, therefore, is not useful as an
antibiotic.

11. Combinations of Clavulanate Potassium
Amoxicillin and the potassium salt of clavulanic acid (Augmentin)
 various fixed-dose oral dosage forms
 treatment of skin, respiratory, ear, and urinary tract infections caused by β-
lactamase–producing bacterial strains.
 Effective against beta-lactamase–producing strains of S. aureus, E. coli, K.
pneumoniae, Enterobacter, H. influenzae, Moraxella catarrhalis, and
Haemophilus ducreyi, which are resistant to amoxicillin alone.
 The oral bioavailability of amoxicillin and potassium clavulanate is similar.
 Clavulanic acid is acid-stable.
 It cannot undergo penicillanic acid formation because it lacks an amide side
chain.

12. Combinations of Clavulanate Potassium
Ticarcillin & Potassium clavulanate (Timentin).
 combined in a fixed-dose injectable form
 For control of serious infections caused by beta-lactamase–producing bacterial
strains.
 Recommended for septicemia, lower respiratory tract infections, and urinary
tract infections caused by - lactamase–producing Klebsiella spp., E. coli, P.
aeruginosa, and other Pseudomonas spp., Citrobacter spp., Enterobacter spp.,
S. marcescens, and S. aureus.

13. Sulbactam
 Synthetic penicillanic acid sulfone or 1,1-dioxopenicillanic acid.
 Potent inhibitor of S. aureus β-lactamase
 & β–lactamases elaborated by Gram-negative bacilli.
 Has weak intrinsic antibacterial activity but potentiates the activity of ampicillin
and carbenicillin against β-lactamase–producing S. aureus and members of
the Enterobacteriaceae family.
 It does not, however, synergize with either carbenicillin or ticarcillin against P.
aeruginosa strains resistant to these agents.
 Failure of sulbactam to penetrate the cell envelope is a possible explanation
for the lack of synergy.

14. Tazobactam
 Tazobactam is also penicillanic acid sulfone.
 More potent β–lactamase inhibitor than sulbactam
 Has a slightly broader spectrum of activity than clavulanic acid.
 It has very weak antibacterial activity.
 Tazobactam is available in fixed-dose, injectable combinations with
piperacillin, (8:1 ratio piperacillin sodium:tazobactam sodium w/w Zosyn)

15. Tazobactam
Zosyn (piperacillin sodium:tazobactam sodium )
 The pharmacokinetics of the two drugs are very similar.
 Both have short half-lives (t1/2 1 hour),
 are minimally protein bound,
 experience very little metabolism, and are
 excreted in active forms in the urine in high concentrations.
 Used in treatment of
 appendicitis,
 postpartum endometritis, and
 pelvic inflammatory disease caused by beta-lactamase–producing E. coli and
Bacteroides spp.,
 skin and skin structure infections caused by beta-lactamase–producing S. aureus,
 pneumonia caused by beta-lactamase–producing strains of H. influenzae.

16. Carbapenems
Class II Inhibitors

17. Carbapenems
Structural modifications
 The sulfur atom is not part of the 5-membered ring but, rather, has been
replaced by a methylene moiety at that position.
 Carbon is roughly half the molecular size of sulfur.
 Consequently, the carbapenem ring system is highly strained and very
susceptible to reactions cleaving the β-lactam bond.
 The sulfur atom is now attached to C-3 as par t of a functionalized side chain
 At C-6, there is a 2-hydroxyethyl group attached with α-stereochemistry.
 Thus, the absolute stereochemistry of the molecule is 5R,6S,8S.
 The endocyclic olefinic linkage also enhances the reactivity of the β-lactam
ring.

18. Carbapenems
Effects on Activity
 Has extremely intense and broad-spectrum antimicrobial activity as
well as
 Inactivates β-lactamases
 Hence molecule has the functional features of the best of the β-
lactam antibiotics as well as the β-lactamase inhibitors.
 Consequently, the carbapenem ring system is highly strained and
very susceptible to reactions cleaving the β-lactam bond.

19. Thienamycin
 first of the carbapenems,
 Isolated from Streptomyces cattleya.
 The terminal amino group in the side chain attached to C-3 is nucleophilic and
attacks the β-lactam bond of a nearby molecule through an intermolecular
react ion destroying activity
 Ultimately, this problem was overcome by changing the amino group to a less
nucleophilic N-formiminoyl moiety by a semisynthetic process to produce
imipenem.
 With these striking differences from the penicillins and cephalosporins, it is
not surprising that thienamycin analogues bind differently to the penicillin
biding proteins (especially strongly to PBP-2), but i t is gratifying that the
result is very potent broad-spectrum activity.

20. Imipenem
 Penetrates very well through porins and is very stable,
 Imipenem is very stable to most –lactamases however Inhibitory to many β-
lactamases.
 Imipenem is not orally active.
 Renal dehydropeptidase-l hydrolyzes imipenem through hydrolysis of the β-lactam
& deactivates.
 An inhibitor for this enzyme, cilastatin, is coadministered with imipenem to protect
it.
 Inhibition of human dehydropeptidase does not have deleterious consequences
hence combination is highly efficacious. On injection, it penetrates well into most
tissues, but not cerebrospinal fluid, and it is subsequently excreted in the urine.
This very potent combination is especially useful for treatment of serious infections
by aerobic gram-negative bacilli, anaerobes, and Staphylococcus aureus.
 It is used clinically for a number of significant infections.

21. Imipenem
 The more common adverse effects are irritation at the infusion site, nausea,
vomiting, diarrhea, and pruritus.
 Of greater concern is the ability of imipenem to induce seizures.
 The risk factors for seizure development include impaired renal function,
preexisting CNS disease or infection, and use of large doses.
 Imipenem is indicated for the treatment of a wide variety of bacterial infections of
the skin and tissues, lower respiratory tract, bones and joints, and genitourinary
tract, as well as of septicemia and endocarditis caused by -lactamase–producing
strains of susceptible bacteria.

22. Meropenem
 II generation, orally inactive - most extensive clinical evaluation.
 Active against multiply-resistant bacteria and for empirical therapy for serious
infections, (bacterial meningitis, septicemia, pneumonia, and peritonitis)
 It exhibits greater potency against Gram-negative and anaerobic bacteria than does
imipenem, but it is slightly less active against most Gram-positive species.
 It is not effective against MRSA.
 Meropenem is not hydrolyzed by DHP-I and is resistant to most -lactamases, including
a few carbapenemases that hydrolyze carbapenem.
 Metabolism : Approx unchanged 70% to 80% - excreted in the urine (IV/IM).
 The remainder is the inactive metabolite formed by hydrolytic cleavage of the beta–
lactam ring.
 It has low incidence of nephrotoxicity due to greater stability to DHP-I & also absence
of the DHP-I inhibitor cilastatin)
 Meropenem appears to be less epileptogenic hence used in the treatment of bacterial

23. Biapenem
 Newer second-generation carbapenem
 Chemical and microbiological properties similar to those of meropenem.
 broad-spectrum antibacterial activity
 Includes most aerobic Gram-negative and Grampositive bacteria and
anaerobes.
 Biapenem is stable to DHP-I and resistant to most -lactamases.
 It is claimed to be less susceptible to metallo-beta-lactamases than either
imipenem or meropenem.
 It is not active orally.

24. Doripenem
 Doripenem is the newest of the approved carbapenems.
 It also contains the 4-β-methyl group, which confers stability toward
dehydropeptidase-1, so it is given as a single agent.
 It is similar in spectrum to imipenem and meropenem but is considered
more potent against Pseudomonas species.

25. Ertapenem
 Ertapenem is another synthetic carbapenem with a rather complex side
chain at C-3.
 As with meropenem and doripenem, the 4-β-methyl group confers
stability toward dehydropeptidase-1.
 It is not active against Pseudomonas or Acinetobacter
 This class of antimicrobial agents is under intensive investigation, and
several analogs are currently in various phases of preclinical
investigation, including tebipenem, an oral prodrug carbapenem.