Principles of Antimicrobial therapyPHRM306

PHARMACOLOGY II

Principles of Antimicrobial TherapyI. Overview:

Antimicrobial therapy takes advantage of the biochemical differences that exist between microorganisms are human beings.

Antimicrobial drugs are effective in the treatment of infections because of their selective toxicity.

That is, they have the ability to injure kill an invading microorganism without harming the cells of the host.

II. Selection of Antimicrobial Agents

1. The organism’s identity2. Its susceptibility to a particular agent3. The site of the infection4. Patient factors5. The safety of the agent 6. The cost of the therapy

A. Identification of the infecting organism

• Characterization of the organism is central to selection of the proper drug.

• A rapid assessment of the nature of the pathogen can sometimes be made on the basis of the Gram stain, which is particularly useful in identifying the presence and morphologic features of microorganisms in body fluids that are normally sterile.

B. Empiric therapy prior to identification of the organism

1. The acutely ill patient with infections of unknown origin

2. Selection a Drug

C. Determination of antimicrobial susceptibility of infective organisms

1. Bacteriostatic drugs: Which arrest the growth & replication of bacteria at serum levels achievable in the patient.

Bactericidal agents: Which kills bacteria at serum levels achievable in patients.

• Cholarmphenicol is static against gram negative rods and is cidal against other organisms such as S. pneumoniae

• 2. Minimum inhibitory concentration: Minimum Inhibitory Concentration (MIC) is the lowest concentration of antibiotics that inhibits bacterial growth.

• To provide effective antimicrobial therapy, the clinically obtainable antibiotic concentration in body fluid should be greater then the MIC.

• 3. Minimum Bactericidal concentration: the minimum bactericidal concentration (MBC) is the lowest concentration of antimicrobial agent that results in a 99.9 percent decline in colony count after overnight broth dilution incubations.

D. Effect of the site of injection on therapy

• The blood Brain Barrier: this barrier is formed by the single layer of tail-like endothelial cells fused by tight junctions that impede entry from the blood to the brain of virtually all molecules, except those that are small and lipophilic.

• The penetration and concentration of an antibacterial agent in the CSF is particularly influenced by the following:

1. Lipid soluble drug, such as quinolones and metronidazole, have significant penetration into the CNS.

• In contrast, β-lactum antibiotics, such as penicillin, are ionized at physiologic PH and have low solubility in lipids. They therefore have limited penetration through the intact blood brain barrier under normal circumstances.

2. Molecular Weight of the drug

3. Protein binding of the drug

E. Patient factors1. Immune System2. Renal Dysfunction: serum creatinine levels

are frequently used as an index of renal function for adjustment of drug regimens.

3. Hepatic dysfunction 4. Poor perfusion5. Age6. Pregnancy7. Lactation F. Safety of the agentG. Cost of the therapy

III. Route of Administration

• Some antibiotics, such as Vancomycin, the aminoglycosides and amphotericin are so poorly absorbed from gastrointestinal tract that adequate serum levels can not be obtained by oral administration.

• Parenteral administration is used for drugs that are poorly absorbed from the gastrointestinal tract and for the treatment of the patients with serious infections.

IV. Determinants of Rational Dosing

• Two important pharmacodynamic properties that have a significant influence on the frequency dosing are:

1.Concentration-depending Killing

2.Post-antibiotic effect

V. Agents used bacterial infections

• Penicillin• Cephalosporin's• Tetracycline's • Aminoglycosides• Macrolides• Fluoroquinolones• Others

VI. Chemotherapeutic Spectra

A. Narrow-Spectrum antibiotics: Isoniazid is active only against mycobacteria

B. Extended-Spectrum: Ampicillin acts against gram positive and some gram negative bacteria

C. Broad-Spectrum Antibiotics: Tetracycline and chloramphenicol affect a wide variety of microbial species

VII. Combinations of antimicrobial drugs

• Treatment of tuberculosis• Advantages of drug combinations: When

infection is of unknown origin. Beta lactums and aminoglycosides show synergism

• Disadvantages of drug combinations: A number of antibiotics act only when organisms are multiplying. Thus co administration of an agent that causes bacteriostasis plus a second agent that is bactericidal may result in the first drug interferring with the action of second.

VIII. Drug Resistance

A. Genetic alterations leading to drug resistance1. Spontaneous mutations of DNA: Emergence of

rifampin-resistant Mycobacterial tuberculosis when rifampin is used as a single antibitotic.

2. DNA transfer of drug resistance

B. Altered expression of proteins in drug-resistant organisms

1. Modification of target site2. Decreased accumulation3. Enzymic Inactivation

IX. Prophylactic Antibiotics

X. Complications of Antibiotics Therapy1.Hypersensitivity2.Direct toxicity3.Superinfections

XI. Sites of Antimicrobial Actions

Beta-Lactam Antibiotics

Introduction

β-Lactam antibiotics are the most widely produced and used antibacterial drugs in the world, and have been ever since their initial clinical trials in 1941.

β-Lactams are divided into several classes based on their structure and function; and are often named by their origin, but all classes have a common β-Lactam ring structure.

N

O

Discovery of Penicillin

• First discovered in 1928 by British physician Alexander Fleming

• Accidental discovery of a contaminated bacterial culture

• Fungus Penicillium notatum killed the culture of Staphylococcus aureus growing in the petri dish

Sir Alexander Fleming

Fleming’s Petri Dish

Zone of Inhibition• Around the fungal colony is a

clear zone where no bacteria are growing

• Zone of inhibition due to the diffusion of a substance with antibiotic properties from the fungus

Penicillin Today

• Still the most widely used antibiotic• Still the drug of choice to treat many bacterial

infections• Scientists have continued to improve the yield

of the drug• Present day strains of P. chrysogenum are

biochemical mutants that produce 10,000 times more penicillin than Fleming's original isolate

Mechanism of ActionTarget- Cell Wall Synthesis

The bacterial cell wall is a cross linked polymer called peptidoglycan which allows a bacteria to maintain its shape despite the internal turgor pressure caused by osmotic pressure differences.

If the peptidoglycan fails to crosslink the cell wall will lose its strength which results in cell lysis.

All β-lactams disrupt the synthesis of the bacterial cell wall by interfering with the transpeptidase which catalyzes the cross linking process.

Peptidoglycan

Peptidoglycan is a carbohydrate composed of alternating units of NAMA and NAGA.

The NAMA units have a peptide side chain which can be cross linked from the L-Lys residue to the terminal D-Ala-D-Ala link on a neighboring NAMA unit.

This is done directly in Gram (-) bacteria and via a pentaglycine bridge on the L-lysine residue in Gram (+) bacteria.

Mechanism

Transpeptidase- PBP

The cross linking reaction is catalyzed by a class of transpeptidases known as penicillin binding proteins. A critical part of the process is the recognition of the D-Ala-D-Ala sequence of the NAMA peptide side chain by the PBP. Interfering with this recognition disrupts the cell wall synthesis. β-lactams mimic the structure of the D-Ala-D-Ala link and bind to the active site of PBPs, disrupting the cross-linking process.

Mechanism of β-Lactam DrugsThe amide of the β-lactam ring is unusually

reactive due to ring strain and a conformational arrangement which does not allow the lone pair of the nitrogen to interact with the double bond of the carbonyl.

β-Lactams acylate the hydroxyl group on the serine residue of PBP active site in an irreversible manner.

Mechanism of β-Lactam Drugs

The hydroxyl attacks the amide and forms a tetrahedral intermediate.

N

O

RS

Me

Me

COOHSER

OH

H

Mechanism of β-Lactam Drugs

The tetrahedral intermediate collapses, the amide bond is broken, and the nitrogen is reduced.

N

RS

Me

Me

COOHSER

O

H

O

Mechanism of β-Lactam Drugs

The PBP is now covalently bound by the drug and cannot perform the cross linking action.

HN

RS

Me

Me

COOHSER

O

O

Bacterial Resistance

Bacteria have many methods with which to combat the effects of β-lactam type drugs.

• Intrinsic defenses such as efflux pumps can remove the β-lactams from the cell.

• β-Lactamases are enzymes which hydrolyze the amide bond of the β-lactam ring, rendering the drug useless.

• Bacteria may acquire resistance through mutation at the genes which control production of PBPs, altering the active site and binding affinity for the β-lactam .

Range of Activity

β-Lactams can easily penetrate Gram (+) bacteria, but the outer cell membrane of Gram (-) bacteria prevents diffusion of the drug. β-Lactams can be modified to make use of import porins in the cell membrane.

β-Lactams also have difficulty penetrating human cell membranes, making them ineffective against atypical bacteria which inhabit human cells.

Any bacteria which lack peptidoglycan in their cell wall will not be affected by β-lactams.

Toxicity

β-Lactams target PBPs exclusively, and because human cell membranes do not have this type of protein β-lactams are relatively non toxic compared to other drugs which target common structures such as ribosomes.

About 10% of the population is allergic (sometimes severely) to some penicillin type β-lactams.

Classes of β-Lactams

The classes of β-lactams are distinguished by the variation in the ring adjoining the β-lactam ring and the side chain at the α position.

Penicillin

N

O

HN

SMe

Me

COOH

C

R

O

Modification of β-Lactams

β-Lactam type antibiotics can be modified at various positions to improve their ability to:

-be administered orally (survive acidic conditions)-be tolerated by the patient (allergies)-penetrate the outer membrane of Gram (-) bacteria-prevent hydrolysis by β-lactamases-acylate the PBPs of resistant species (there are many

different PBPs)

Penicillins- NaturalNatural penicillins are those which can be obtained

directly from the penicillium mold and do not require further modification. Many species of bacteria are now resistant to these penicillins.

Penicillin G

not orally active

N

O

HN

SMe

Me

COOH

C

O

Penicillin G in Acidic Conditions

Penicillin G could not be administered orally due to the acidic conditions of the stomach.

N

O

NS

Me

Me

COOH

C

O

H

H+

Penicillin V

Penicillin V is produced when phenoxyacetic acid rather than phenylacetic acid is introduced to the penicillium culture. Adding the oxygen decreases the nucleophilicity of the carbonyl group, making penicillin V acid stable and orally viable.

N

O

HN

SMe

Me

COOH

C

O

O

Ph

ProductionAll commercially available β-lactams are initially produced

through the fermentation of bacteria.Bacteria assemble the penicillin molecule from L-AAA, L-

valine, and L-cysteine in three steps using ACV (L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine) synthase, IPN (Isopenicillin N ) synthase, and acyltransferase.

Modern recombinant genetic techniques have allowed the over expression of the genes which code for these three enzymes, allowing much greater yields of penicillin than in the past.

Penicillin Biosynthetic Pathway

o

Semi-Synthetic Penicillins

The acyl side chain of the penicillin molecule can be cleaved using enzyme or chemical methods to produce 6-APA, which can further be used to produce semi-synthetic penicillins or cephalosporins

75% of the penicillin produced is modified in this manner.

N

O

H2NS

Me

Me

COOH

OHR

O

Penicillins- Antistaphylococcal

Penicillins which have bulky side groups can block the β-Lactamases which hydrolyze the lactam ring.

N

O

NHS

Me

Me

COOH

O

Penicillins- Antistaphylococcal

These lactamases are prevalent in S. aureus and S. epidermidis, and render them resistant to Penicillin G and V. This necessitated the development of semi-synthetic penicillins through rational drug design.

Methicillin was the first penicillin developed with this type of modification, and since then all bacteria which are resistant to any type of penicillin are designated as methicillin resistant. (MRSA- methicillin-resistant S. aureus)

Penicillins- AntistaphylococcalMethicillin is acid sensitive and has been

improved upon by adding electron withdrawing groups, as was done in penicillin V, resulting in drugs such as oxacillin and nafcillin.

Due to the bulky side group, all of the antistaphylococcal drugs have difficulty penetrating the cell membrane and are less effective than other penicillins.

Oxacillin

Nafcillin

Penicillins- AminopenicillinsIn order to increase the range of activity, the penicillin

has been modified to have more hydrophilic groups, allowing the drug to penetrate into Gram (-) bacteria via the porins.

Ampicillin R=Ph

Amoxicillin R= Ph-OH N

O

HN

SMe

Me

COOH

C

C

R

O

NH2H

Penicillins- Aminopenicillins

These penicillins have a wider range of activity than natural or antistaphylococcal drugs, but without the bulky side groups are once again susceptible to attack by β-lactamases

The additional hydrophilic groups make penetration of the gut wall difficult.

Penicillins- Aminopenicillins

Due to the effectiveness of the aminopenicillins, a second modification is made to the drug at the carboxyl group.

Changing the carboxyl group to an ester allows the drug to penetrate the gut wall where it is later hydrolyzed into the more polar active form by esterase enzymes.

This has greatly expanded the oral availability of the aminopenicillin class.

Penicillins- Extended Spectrum

Extended spectrum penicillins are similar to the aminopenicillins in structure but have either a carboxyl group or urea group instead of the amine

N

O

HN

SMe

Me

COOH

C

C

R

O

CO2RH

N

O

HN

SMe

Me

COOH

CR

O

NHR2N

O

Penicillins- Extended Spectrum

Like the aminopenicillins the extended spectrum drugs have an increased activity against Gram (-) bacteria by way of the import porins.

These drugs also have difficulty penetrating the gut wall and must be administered intravenously if not available as a prodrug.

These are more effective than the aminopenicillins and not as susceptible to β-lactamases

Cephalosporins

• Cephalosporins were discovered shortly after penicillin entered into widespread product,

• First discovered in 1945 from a Cephalosporium fungi

• but not developed till the 1960’s.

Chemical structure of cephalosporins

• Cephalosporins are similar to penicillins but have a 6 member dihydrothiazine ring instead of a 5 member thiazolidine ring.

• Derived from 7-aminocephalosporanic acid. 7-aminocephalosporanic acid (7-ACA) can be obtained from bacteria, but it is easier to expand the ring system of 7-APA because it is so widely produced.

• They suffer the “attack” of bacteria at their beta-lactam ring.

Cephalosporins

Unlike penicillin, cephalosporins have two side chains which can be easily modified. Cephalosporins are also more difficult for β-lactamases to hydrolyze.

N

O

HN

C

R

O

S

CO2H

OAc

Mechanism of ActionMechanism of Action

NO

HHHNR

O

S

CO2H

OC

Me

O

7

OH

Ser Enzyme

-CH3CO2-

NO

HHHNR

O

S

CO2HO

Ser

Enzyme

NoteNoteThe acetoxy group acts as a good leaving group and aids the mechanismThe acetoxy group acts as a good leaving group and aids the mechanism

Cephalosporins, Classification

Cephalosporins are divided into four generations with original agents being referred to as first-generation cephalosporins, and the most recent agents as fourth-generation cephalosporins

In general, the spectrum of activity of cephalosporins increases with each generation because of decreasing susceptibility to bacterial -lactamases

First-Generation Cephalosporins Examples: cephradine, cephalexin, cephadroxil, cephapirin

They are active against most staphylococci, pneumococci, and all streptococci, with the important exception of enterococci

Their activity against aerobic G-ve bacteria and against anaerobes is limited

They act as penicillin G substitutes. They are resistant to -lactamase

They distribute widely throughout the body, but do not penetrate well into the CSF (not used for meningitis)

They should not be given to patients with a history of immediate-type hypersensitivity reactions to penicillins

Second-Generation Cephalosporins They have a broader bacteriologic

spectrum than do the first-generation agents

They are more resistant to -lactamase than the first-generation drugs

For example, cefamandole, cefuroxime, and cefaclor not only are more active against G-ve enteric bacteria but are active against both -lactamase-negative and -positive strains of H. influenzae

Excretion is primarily renal, and they distribute widely. However, they do not attain sufficient concentrations in the CSF to warrant their use in the treatment of bacterial meningitis

Third-Generation Cephalosporins These agents retain much of the G+ve activity of the

first two generations, although their anti-staphylococcal activity is reduced. They are remarkably active against most G-ve enteric isolates

Some third-generation cephalosporins (e.g., ceftazidime and cefoperazone) also are active against most isolates of P. aeruginosa

These antibiotics diffuse well into most tissues (e.g., cefotaxime and ceftriaxone

Indications include suspected bacterial meningitis and treatment of hospital-acquired multiple-resistant G-ve aerobic infections and suspected infections in certain compromised hosts

Ceftriaxone is the drug of choice in treating infections caused by N. gonorrhoeae in geographic areas with a high incidence of penicillin-resistant isolates

Cephalosporins, Classification

Fourth-Generation Cephalosporins This generation of cephalosporins (such as cefepime, cefpirome) combines the anti-

staphylococcal activity (but only those that are methicillin-susceptible) of first-generation agents with the G-ve spectrum (including Pseudomonas) of third-generation cephalosporins

This class of cephalosporins have increased stability against hydrolysis by -lactamases

Cephalosporins, Classification

These drugs are usually administrated parenterally

They demonstrated good penetration into CSF compared to cefotaxime

They are highly active against common pediatric meningeal pathogens, including Streptococcus pneumoniae. Their in vitro activity against penicillin-resistant pneumococci is generally twice that of cefotaxime or ceftriaxone

Fourth-generation agents are particularly useful for the empirical treatment of serious infections in hospitalized patients when gram-positive microorganisms, Enterobacteriaceae, and Pseudomonas all are potential etiologies

Fifth-Generation Cephalosporins

These are novel cephalosporins with activity against MRSA

Example: Ceftaroline

Ceftaroline, the active metabolite of a N-phosphono prodrug, ceftaroline fosamil, has been recently approved by the US FDA for the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia

This antimicrobial agent binds to penicillin binding proteins (PBP) inhibiting cell wall synthesis and has a high affinity for PBP2a, which is associated with methicillin resistance

Ceftaroline is consistently active against multidrug-resistant Streptococcus pneumoniae and Staphylococcus aureus, including methicillin-resistant strains

Cephalosporins, Classification

Carbapenems

Carbapenems are a potent class of β-lactams which attack a wide range of PBPs, have low toxicity, and are much more resistant to β-lactamases than the penicillins or cephalosporins.

N

O

S

CO2H

NH2

Me

HO

H

Carbapenems

Thienamycin, discovered by Merck in the late 1970’s, is one of the most broad spectrum antibiotics ever discovered.

It uses import porins unavailable to other β-lactams to enter Gram (-) bacteria.

Due to its highly unstable nature this drug and its derivatives are created through synthesis, not bacterial fermentation.

Carbapenems

Thienamycin was slightly modified and marked as Imipenem. Due to its rapid degradation by renal peptidase it is administered with an inhibitor called cilastatin under the name Primaxin. Imipenem may cause seizures or sever allergic reactions.

Other modifications of Thienamycin have produced superior carbapenems called Meropenem and Ertapenem, which are not as easily degraded by renal peptidase and do not have the side effects of Imipenem.

Monobactams have a monocyclic β-lactam ring and are resistant to β-lactamases.

Aztreonam was isolated from Chromobacterium violaceum .

Aztreonam is the first clinically useful monobactam.

The antimicrobial activity of Aztreonam differs from those of other β-lactam antibiotics and more closely resembles that of an aminoglycosides in activity without the nephrotoxicity of aminoglycosides.

Aztreonam

Clinical uses of aztreonam

Active against G- aerobes onlyAlternative for penicillins ( piperacillin ) and

cephalosporins ( ceftazidime ) allergic pts for G- infections

Safe alternative to aminoglycosides, esp. in elderly and pts with renal impairements

β-Lactamases

β-Lactamases were first discovered in 1940 and originally named penicillinases.

These enzymes hydrolyze the β-lactam ring, deactivating the drug, but are not covalently bound to the drug as PBPs are.

Especially prevalent in Gram (-) bacteria.Three classes (A,C,D) catalyze the reaction using a

serine residue, the B class of metallo- β-lactamases catalyze the reaction using zinc.

β-Lactamase Inhibitors

There are currently three clinically available β-lactamase inhibitors which are combined with β-lactams; all are produced through fermentation.

These molecules bind irreversibly to β-lactamases but do not have good activity against PBPs. The rings are modified to break open after acylating the enzyme.

N

O

S

CO2H

OO

N

O

O

CO2H

OH

β-Lactam/Inhibitor combinations

Aminopenicillins:ampicillin-sulbactam = Unasynamoxicillin-clavulante = Augmentin

Extended-Spectrum Penicillinspiperacillin-tazobactam = Zosynticarcillin-clavulanate = Timentin