Chemotherapeutic Agents

Definitions
Chemotherapeutic agent: any chemical used to treat disease
Antimicrobial agent: any chemical used to treat infection Antibiotic: compound produced by one microorganism that kills/inhibits another Antiseptic: use antimicrobial that is too toxic for internal

Disinfectant: chemical so toxic that it is used only on inanimate objects

The Golden Rule

The right drug for the right bug

Ideal Antimicrobial Agent

Soluble in body Stable in body Selectively toxic Consistent Toxicity Non-allergic Bacterial resistance difficult to develop Long shelf life Reasonable cost

Antibiotic
a chemical substance produced by certain molds and bacteria that kills or inhibits the growth of another microorganism Bacteriostatic inhibits Bacteriocidal kills Altough initially isolated as natural products of microorganims, can now also be synthesized in the lab

Antibiotic producers
Fungi Bacillus species Actinomycetes

Discovery of Penicillin
Flemming
in 1928 observed inhibition of Staphylococcus by Penicillium mold

1930s
Clinical trials of penicillin
S. aureus Penicillium

1940s
Developed mass production of penicillin due to need for it in World War II

Bacillus sp.
Bacillus sp.
Gram-positive Spores-formers

Streptomyces
member of the bacterial order Actinomycetales most successful genus in Streptomyces Over 500 species Actinomycetes Gram-positive bacteria. resemble fungi, but are true bacteria prokaryotic cells unlike eukaryotic fungal cells. non-motile, filamentous form spores from aerial filaments distinct from bacterial endospores. Produce numerous antibiotics Streptomyces species are found worldwide in soil

Terms/Concepts of Antimicrobial Agent Selective Toxicity Spectrum of Activity Mode of Action Side Effects Resistance

Selective Toxicity
Concentration that eliminates pathogen
Therapeutic dosage level

Concentration that causes damage to host

Toxic dosage level

Chemotherapeutic index =
Maximum tolerable dose (per Kg body weight) Minimum therapeutic dose (per Kg body weight)
__

High for Penicillin (bacteria) low for heavy metal compounds (worm parasite)

Spectrum of Activity
Range of microorganisms that are affected by agent Broad spectrum
Wide range, e.g. both gram-pos & gram-neg Used when infective bacterial agent on is not precisely identified

Narrow spectrum
Limited number, or specific group of bacteria Used to prevent development of resistance Less of an affect on normal bacterial flora

Antibiotic Spectrum

Obligate intracellular microorganisms

Chlamydia tiny, non-motile, spherical bacteria Rickettsia small, non-motile, gram-negative bacteria

Antibiotic Mode of Action

1. 2. 3. 4. 5.

Cell Wall Cell membrane Protein synthesis Nucleic Acid Synthesis Antimetabolites

Some clinically important antibiotics

Antibiotic
Penicillin Bacitracin Polymyxin B Amphotericin B Erythromycin Neomycin Streptomycin Tetracycline Vancomycin Rifamycin

Producer organism
Penicillium chrysogenum Bacillus subtilis Bacillus polymyxa Streptomyces nodosus Streptomyces erythreus Streptomyces fradiae Streptomyces griseus Streptomyces rimosus Streptomyces orientalis Streptomyces mediterranei

Activity
Gram-positive bacteria Gram-positive bacteria Gram-negative bacteria Fungi Gram-positive bacteria Broad spectrum Gram-negative bacteria Broad spectrum Gram-positive bacteria Tuberculosis

Site or mode of action

Wall synthesis Wall synthesis Cell membrane Cell membrane Protein synthesis Protein synthesis Protein synthesis Protein synthesis Protein synthesis Protein synthesis

Cell Wall Disruption

Bacteria have a high internal osmotic pressure Without a sturdy cell wall, this pressure will cause membrane to burst Antibiotics can interfere with formation of the cell wall Results in cell death by cell bursting open

Cell Wall

penicillin

Penicillin has a 4-member ring looks like part of the cell wall to the cross-linking enzyme Penicillin competes with the normal cell wall component for the cross-linking enzyme, i.e. competitive inhibition Prevents this enzyme from cross-linking cell wall

Penicillin
Penicillin G is the natural penicillin Produced by Penicillium notatum Administered by injection because is degraded by stomach acids Rapidly absorbed into blood & rapidly excreted Used against: streptococcus, meningococcus, pneumonococcus, spirochetes, clostridia, aerobic grampositive rods, some staphylococcus and gonococcus Active in urine; so used for urinary tract infections Generally nontoxic Problems Allergic reaction (~5% in adults) Bacterial resistance

Semi-synthetic Penicillins
Add a side-chain to the penicillin structure Alters: Chemical characteristics Spectrum of activity Development of bacterial resistance Methicillin Penicillinase resistant resistance by an different mechanism developed Ampicillin broad spectrum ( gram-neg & gram-pos) acid resistant, i.e. oral administration

Cell Wall - Polypeptide Antibiotics

Bacitracin Produced by Bacillus licheniformis Small polypeptide Inhibits cell wall formation Used on lesions & wounds because: Poorly absorbed in body Toxic to kidneys Vancomycin Streptomyces Very narrow spectrum Used against Staphylococcus that is resistant to penicillin Vancomycin resistance is now developing

Cell Wall - Antimycobacterial

Isoniazid (INH) Inhibits synthesis of mycolic acid in cell wall of Mycobacteria Tuberculosis Administered with other antibiotics to prevent development of resistance Ethambutanol Inhibits incorporation of mycolic acid into cell wall Weak drug just used secondarily to avoid development of resistance to INH Rifampin (inhibits mRNA synthesis) Hits alternative target in cell

 Isoniazid Complications
Competitive inhibitor of Vitamin B6 Prevents enzymes from converting Vitamin B6 to useful molecules Often supplement patients diet with extra Vitamin B6 during treatment

Cell Wall - Antifungals

Echinocandins First new class of antifungals in 40 years Fungal cell walls contain glucans (polysaccharide) Echinocandins inhibit synthesis of glucans So inhibits synthesis of fungal cell wall

Cell Membrane Disruption

Act as detergent Distort cell membrane
Bind to phospholipids

Membrane loses function

Selective transport

Especially effective against gram-negatives

because of their high lipid content in outer membrane

Can adversely affect host cells

Polymyxins
From soil bacterium, Bacillus polymyxa Targets prokaryotic membrane Usually for skin infections by gram-negatives Toxic so used internally only under close medical supervision Kidney damage Respiratory arrest

Gramicidin
Bacillus brevis Gram-positive infections Topical ointment highly toxic cannot be administered internally used only on the skin as a lotion or ointment. Interacts with bacterial membrane to form channel Interferes with membrane function increases the permeability of the bacterial cell membrane

Antifungal antibiotics
Many used for topical application only, due to toxicity for superficial fungal infections only Systemic fungal infections Can be toxic to the animal host Toxicity due to adverse effect on specific tissues kidneys, bone marrow

Amphotericin B
Streptomyces inhibits membrane function of fungi selective transport cell shape binds to sterols in the cell membranes of fungi degradation of membrane integrity and cell lysis Fungal cell membrane contain ergosterol Animal cells contain cholesterol

Inhibit Nucleic Acid Synthesis

DNA synthesis inhibition
Metronidazoles add functional groups to DNA Makes DNA weak and prone to strand breakage antibacterial and antiprotozoal drug Amebiasis, giardiasis, Helicobacterer pylorii (ulcers) Side effects: nausea, vomiting, diarrhea Nalidixic acid & quinolines specifically bind to enzymes necessary for DNA synthesis DNA gyrase, that uncoils and coils the DNA Prevents DNA replication Bacteria die out

Inhibit mRNA synthesis - Transcription Rifampin

Streptomyces binds to bacterial RNA polymerase blocks bacterial DNA from transferring its information to RNA Used in U.S. for TB and meningococcus treatment Can cause liver damage

RNA synthesis disruption - Antifungals

Flucytosine Analogue of cytosine Fungi have enzyme to convert Flucytosine to 5-Flurouracil 5-Flurouracil is incorporated into RNA in place of cytosine Disrupts protein synthesis because: Either incorrect amino acid or no amino acid is put into protein protein synthesis stops

Protein Synthesis - Translation

RNA translation to protein bacterial ribosomes read the mRNA code bring in the tRNA which holds the anti-codons Carries in proper amino acid to add to polypepetide Antimicrobial agents target the large and small subunits of bacterial ribosomes. because ribosomes of prokaryotes are different from those of eukaryotes (70S vs 80S) Therefore specific to prokaryotic ribosomes Less toxic to eukaryotic host

Peptide chain elongation

Streptomycin, kanamycin, tetracycline & neomycin bind to the smaller 30S ribosomal subunit, Chloramphenicol, erythromycin & streptogramins bind to the 50S ribosome Streptogramins used with vancomycin-resistant pathogens Can be toxic to kidney & inner ear (ringing) Tetracycline stains teeth Chloramphenicol can damage bone marrow Aplastic anemia low RBCs & platelets count

Anti-metabolites

PABA

Sulfonilamides, or Sulfa drugs First antimicrobial agent Folic acid analogue (= similar structure) Coenzyme necessary for metabolic pathways Similar structure to a precursor of folic acid Competitive inhibition Sulfanilamide Inhibit synthesis of folic acid Humans require their folic acid in their diet humans do not synthesize folic acid So human metabolism is not inhibited by sulfonilamides Allergies can develop

Antiviral
Purine & pyrimidine analogs Inhibit viral replication The most effective ones are incorporated more rapidly into viral-infected cells than uninfected cells

Purine

vidarabine

pyrimidine

idoxuridine

Acyclovir
a guanosine analog Acyclovir is phosphorylated by viral enzyme becomes a false nucleotide Viral enzyme has a higher affinity for acyclovir than does host enzyme If false nucleotide is used by DNA polymerase will halt DNA synthesis

nucleoside
Nucleoside kinase

Acyclovir and Thymine Kinase

nucleotide
DNA Polymerae

Acyclovir
Nucleoside kinase

Acyclovir phosphate
DNA Polymerae

DNA Synthesis stops

Interferon
Naturally synthesized by infected host cell Prevents surrounding cell from being infected Helps to limit infection

Antiprotozoal
Quinines Malaria; Plasmodium vivax Interfers with protein synthesis Especially red blood cells Prevents parasite from metabolizing hemoglobin as an energy and carbon source Metranidazole causes breakage of DNA strands Trichomonas infections, Giardiasis,

Antihelminthic
Niclosamide
Interfers with carbohydrate metabolism tapeworms

Mebendazole
Interfers with glucose uptake roundworms

Piperazine
Neurotoxin Paralyzes muscles of worm Can cause convulsions pinworms

Side Effects
Toxicity Allergy Disrupt Normal Flora
Superinfection Overgrowth More likely with broad spectrum antibiotics

Resistance

Antibiotic resistance
New enzymes Degrade or alter antibiotic Alter Target cant bind wont work Ribosomes, enzymes Alter membrane Prevent transport in Actively pump out Bypass affected pathway by using an alternative pathway

Destroy Antibiotic

Penicillin resistance
Bacteria obtains gene for production of the enzyme lactamase Breaks covalent bond of the lactam ring of penicillin

Resistance by ribosome alteration

A bacterium alters the antibiotic's target
the 50S ribosomal subunit. The drug is no longer able to bind to the ribosome Cant block protein synthesis.
Normal ribosome

Altered ribosome

Alter antibiotics transport protein

Efflux Mechanism Pump Antibiotic out

Development of Antibiotic Resistance

Transferring Antibiotic Resistance

Increase in Antibiotic Resistance

Antibiotic Resistance

Basis of microbial resistance

Inherent (Natural) Resistance

Streptomycete has gene which is responsible for resistance to own antibiotic Gram-negative outer membrane (permeability) lack of transport system (Xanthomonas) lack of target (structure or biochemical reaction)

Basis of Microbial Resistance

Acquired resistance (previously sensitive)

change in bacterial genome
mutation and selection (vertical evolution)
spontaneous

exchange of genes between strains and species (horizontal evolution)

conjugation transduction transformation

Medical problem of Drug Resistance

development of resistance is correlated with level of antibiotic use

overuse has increased the incidence of and selection for resistance-conferring mutations

Examples of overuse/misuse

prophylactic use before surgery empiric use (no known etiologic agent) increased use of broad spectrum agents antibiotics in animal feeds pediatric use for viral infections patients who do not complete course (TB,AIDS)

Spread of Resistance

day-care, nursing homes, correctional facilities fecal-oral (sanitation, animal feeds) sexual transmission respiratory transmission international travel immunosupression (cancer, organ transplants)

How to control the problem

Use a different antibiotic

we are running out (vancomycin resistant S. aureus)

Modify antibiotic to circumvent resistance

organisms keep catching up

Use antibiotic combinations

double edged sword

Other strategies

reduce consumption of antibiotics

physician education animal feeds

use more targeted antibiotics

broad spectrum, magic bullet is not the answer

patient education - take the entire treatment course

Other Strategies

New Drugs
currently about 100 antibiotics on market three main mechanisms of action
inhibition of protein synthesis inhibition of cell wall synthesis/maintenance inhibition of DNA replication

Other strategies

New drugs
pharmaceutical industry putting resources back into discovery, SAR discoveries in microbial physiology and genetics offering new targets combinatorial chemistry - mass screening liaisons with university researchers starting to show success

New Antibiotics
Cell Wall inhibitors - based on beta lactam skeleton
carbapenems - broad spectrum
imipenem (Primaxin) usually given with cilastatin (prevents degradation by renal enzymes) meropenem (Zeneca) - enhaced anti-Pseudomonas activity

penems - beta lactamase stable

Aztreonam (Bristol-Myers Squibb) Moxalactam (Lilly)

New Antibiotics

New Quinolones - result of SAR

Sparfloxacin (Rhone/Poulenc) Clinafloxacin (Parke-Davis)

Glycylcyclines
derivatives of tetracyclines resistant to efflux mechamisms

New Antibiotics

Drugs to inhibit resistance mechanisms

better beta lactamase inhibitors blockers of tetracycline pump Isoniazid resistance blocker (M.tb)

Drugs which interfere with membranes

knowledge of membrane structure and function increasing

Conclusions Resistance to antimicrobials is currently and will continue to be a problem Safe and effective new antimicrobials are needed now and will continue to be needed New understanding of microbial physiology and genetics is presenting many new targets Pharma resources flowing back into antibiotic development Competition is high

Antibiotics summary

Triad of chemotherapy Selective toxicity Mechanisms of action: post-antibiotic effect Resistance: use big guns cautiously Eliminate misuses, e.g., use of antibacterials Need to discover and develop new antibiotics;
antibiotics in amphibian and human skin infections

for viral